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PubMed2006

Efficacy of lipase from Aspergillus niger as an additive in detergent formulations: a statistical approach.

The efficacy of lipase from Aspergillus niger MTCC 2594 as an additive in laundry detergent formulations was assessed using response surface methodology (RSM). A five-level four-factorial central composite design was chosen to explain the washing protocol with four critical factors, viz. detergent concentration, lipase concentration, buffer pH and washing temperature. The model suggested that all the factors chosen had a significant impact on oil removal and the optimal conditions for the removal of olive oil from cotton fabric were 1.0% detergent, 75 U of lipase, buffer pH of 9.5 and washing temperature of 25 degrees C. Under optimal conditions, the removal of olive oil from cotton fabric was 33 and 17.1% at 25 and 49 degrees C, respectively, in the presence of lipase over treatment with detergent alone. Hence, lipase from A. niger could be effectively used as an additive in detergent formulation for the removal of triglyceride soil both in cold and warm wash conditions.

Aspergillus niger↗
PubMed1997

Altered acyl chain length specificity of Rhizopus delemar lipase through mutagenesis and molecular modeling.

The acyl binding site of Rhizopus delemar prolipase and mature lipase was altered through site-directed mutagenesis to improve lipase specificity for short- or medium-chain length fatty acids. Computer-generated structural models of R. delemar lipase were used in mutant protein design and in the interpretation of the catalytic properties of the resulting recombinant enzymes. Molecular dynamics simulations of the double mutant, val209trp + phe112trp, predicted that the introduction of trp112 and trp209 in the acyl binding groove would sterically hinder the docking of fatty acids longer than butyric acid. Assayed against a mixture of triacylglycerol substrates, the val209trp + phe112trp mature lipase mutant showed an 80-fold increase in the hydrolysis of tributyrin relative to the hydrolysis of tricaprylin while no triolein hydrolysis was detected. By comparison, the val94Trp mutant, predicted to pose steric or geometric constraints for docking fatty acids longer than caprylic acid in the acyl binding groove, resulted in a modest 1.4-fold increase in tricaprylin hydrolysis relative to the hydrolysis of tributyrin. Molecular models of the double mutant phe95asp + phe214arg indicated the creation of a salt bridge between asp95 and arg214 across the distal end of the acyl binding groove. When challenged with a mixture of triacylglycerols, the phe95asp + phe214arg substitutions resulted in an enzyme with 3-fold enhanced relative activity for tricaprylin compared to triolein, suggesting that structural determinants for medium-chain length specificity may reside in the distal end of the acyl binding groove. Attempts to introduce a salt bridge within 8 A of the active site by the double mutation leu146lys + ser115asp destroyed catalytic activity entirely. Similarly, the substitution of polar Gln at the rim of the acyl binding groove for phe112 largely eliminated catalytic activity of the lipase.

Caprylates↗
PubMed1988

Role of lipoprotein lipase activity on lipoprotein metabolism and the fate of circulating triglycerides in pregnancy.

The mechanism that induces maternal hypertriglyceridemia in late normal pregnancy, and its physiologic significance are reviewed as a model of the effects of sex steroids on lipoprotein metabolism. In the pregnant rat, maternal carcass fat content progressively increases up to day 19 of gestation, then declines at day 21. The decline may be explained by the augmented lipolytic activity in adipose tissue that is seen in late pregnancy in the rat. This change causes maternal circulating free fatty acids and glycerol levels to rise. Although the liver is the main receptor organ for these metabolites, liver triglyceride content is reduced. Circulating triglycerides and very-low-density lipoprotein (VLDL)-triglyceride levels are highly augmented in the pregnant rat, indicating that liver-synthesized triglycerides are rapidly released into the circulation. Similar increments in circulating VLDL-triglycerides are seen in pregnant women during the third trimester of gestation. This increase is coincident with a decrease in plasma postheparin lipoprotein lipase activity, indicating a reduced removal of circulating triglycerides by maternal tissues or a redistribution in their use among the different tissues. During late gestation in the rat, tissue lipoprotein lipase activity varies in different directions; it decreases in adipose tissue, the liver, and to a smaller extent the heart, but increases in placental and mammary gland tissue. These changes play an important role in the fate of circulating triglycerides, which are diverted from uptake by adipose tissue to uptake by the mammary gland for milk synthesis, and probably by the placenta for hydrolysis and transfer of released nonesterified fatty acids to the fetus. After 24 hours of starvation, lipoprotein lipase activity in the liver greatly increases in the rat in late pregnancy; this change is not seen in virgin animals. This alteration is similar to that seen in liver triglyceride content and plasma ketone body concentration in the fasted pregnant rat. In the fasting condition during late gestation, heightened lipoprotein lipase activity is the proposed mechanism through which the liver becomes an acceptor of circulating triglycerides, allowing their use as ketogenic substrates, so that both maternal and fetal tissues may indirectly benefit from maternal hypertriglyceridemia. Changes in the magnitude and direction of lipoprotein lipase activity in different tissues during gestation actively contribute both to the development of hypertriglyceridemia and to the metabolic fate of circulating triglycerides.(ABSTRACT TRUNCATED AT 400 WORDS)

Adipose Tissue↗
PubMed1983

Rapid preparation of a relatively stable, partially purified lipoprotein lipase from perfused rat heart.

Rat hearts, extensively washed with cold 0.15 M NaCl solution, were perfused with 5 ml of 0.15 M NaCl containing 16 U of heparin and 10% glycerol to release endothelium-bound lipoprotein lipase. Approximately 100 mU of enzyme activity could be released from each heart (weighing about 1.7 g). Several hearts could be sequentially perfused with the same heparin solution to enrich it in lipase activity. When compared with other equally rapid and frequently used sources of rat lipoprotein lipase (such as heart acetone powder or postheparin plasma), our enzyme preparation had a much higher specific activity suggesting that a greater purification level had been already achieved in a single step. In addition, this lipoprotein lipase preparation contained only trace amounts of lipids, was stable for an hour at 37 degrees C and retained 75% of its activity after 10 days at 4 degrees C. The described procedure is a quick way to prepare a soluble, partially purified and relatively stable lipoprotein lipase that may be useful especially for the in vitro preparation of triacylglycerol-rich lipoprotein remnants.

Animals↗
PubMed1988

Triacylglycerol lipase-mediated release of arachidonic acid for renal medullary prostaglandin synthesis.

The effect of triacylglycerol lipase or triarachidonin on the synthesis of prostaglandin E2 in rabbit kidney medulla slices was examined. Prostaglandin E2 generation was enhanced by exogenous triacylglycerol lipase, indicating that arachidonic acid released from medullary triacylglycerols is readily available for prostaglandin biosynthesis. Triarachidonin stimulated prostaglandin E2 production in a dose-dependent manner. Moreover, the kidney medulla slices showed triacylglycerol lipase activity using trimargarin as substrate. The addition of mepacrine made the stimulatory effect of triarachidonin on prostaglandin E2 formation more pronounced. Studies utilizing EGTA and p-bromophenacyl bromide revealed that triacylglycerol lipase of kidney medulla is independent of Ca2+ and sensitive to a sulfhydryl inhibitor. These results suggest the presence of triacylglycerol lipase and triacylglycerol as a possible candidate for providing free arachidonic acid to cyclooxygenase in kidney medulla.

5,8,11,14-Eicosatetraynoic Acid↗
PubMed1975

Interference with the transport of heparin-releasable lipoprotein lipase in the perfused rat heart by colchicine and vinblastine.

The effect of pretreatment with colchicine or vinblastine on the lipoprotein lipase activity of rat heart was studied. Administration of colchicine or vinblastine 4 h prior to perfusion of the heart caused a very marked reduction in lipoprotein lipase activity released into the perfusate within 1 min of heparin perfusion. At the same time an increase in residual heart lipase occurred so that total lipoprotein lipase content of the heart (heparin releasable plus residual) did not change. The colchicine effect was dose and time dependent; no decrease in heparin-releasable enzyme activity occurred after only 30 min of pretreatment or upon addition of colchicine into the perfusate. These results indicate that colchicine did not impede enzyme synthesis or its release from the cell surface, but may have interfered with the transport of lipoprotein lipase from the site of its synthesis to the endothelial cell surface.

Animals↗
PubMed1977

Partial purification and characterization of a triacyglycerol lipase from rat liver cytosol.

A triacyglycerol lipase (EC 3.1.1.3) was purifiec about 60-fold from rat liver cytosol by delipidation with acetone and ethyl ether, hydroxyapatitie and Sephadex G-100 column chromatographies and isoelectrofocusing electrophoresis. The partially purified enzyme had a molecular weight of approximately 42 000 and an isolectric point of 7.2. The Km for trioleylglycerol was 0.33 mM and the pH optimum was around 8.0. The activity of the enzyme was not dependent on serum lipoproteins, but was stimulated about 2-fold by several proteins such as serum albumin, lipoproteins, gamma-globulin and ovalbumin. The lipase hydrolyzed trioleyglycerol to oleic acid and glycerol. NaCl had no effect on the enzymatic activity. Some physical and kinetic properties of the partially purified lipid-free lipase were different from those of crude non-delipidated lipase and also from those of a neutral triacylglycerol lipase which was recently purified partially from pig liver cytosol (Ledford, J.H. and Alaupovic, P. (1975) Biochim. Biophys. Acta 398, 132-148).

Animals↗
PubMed1978

Determination of lipoprotein-lipase activity in human skeletal muscle tissue.

An in vitro assay system was developed for the determination of lipoprotein-lipase activity in 10--30-mg specimens of human skeletal muscle tissue. The reaction medium of the assay was based on a glycine buffer of pH 8.3 (at 37 degrees C) with a heparin concentration of 1.5 g/l (about 180 IU/ml). The enzyme activity was measured as the release of [3H]oleic acid from a serum-activated, triglyceride emulsion, in which [3H]trioleate was used as trace substance. The enzyme activity studied had the characteristic properties of lipoprotein-lipase activity, i.e. it was activated by the addition of serum or apolipoprotein C-II and inhibited in the presence of high ionic strength, protamine sulphate or apolipoprotein C-III. A mean Km of 0.40 +/- 0.13 (S.D.) mmol/l for triglyceride substrate was found in tissue samples that had very different concentrations of lipoprotein-lipase activity. This Km was similar to the low fasting concentrations of very low density lipoprotein triglycerides often found in healthy individuals. The lipoprotein-lipase activity was not decreased freezing and storing the tissue specimens in liquid nitrogen. The within-day variation of the method was 16 percent and the between-day variation 8 percent. Muscle tissue from the vastus lateralis muscle had, on the average, a 60 percent higher concentration of lipoprotein-lipase activity than the rectus abdominis muscle in the same subject.

Adipose Tissue↗
PubMed1978

Lipase-colipase interactions during gel filtration. High and low affinity binding situations.

The interaction of porcine pancreatic lipase and colipase was studied during gel filtration in columns eluted with a variety of buffers. High and low affinity binding situations were observed under different conditions. Low affinity binding could only be detected at the high lipase-colipase concentrations encountered during batch purification (10(-3)-10(-4) M). Even in this situation the rapid dissociation of the weak complex during filtration resulted in considerable separation of the two proteins. High affinity binding of lipase to colipase was observed at protein eluant concentrations as low as 10(-8) M on columns equilibrated with oleic acid-taurodeoxycholate mixed micelles. This binding did not take place on columns equilibrated with simple bile salt and mixed phosphatidylcholine-cholesterol-bile salt micelles. Colipase alone exhibited strong binding to phosphatidylcholine and fatty acid mixed bile salt micelles when applied together in a sample on columns eluted with pure bile salt micelles, lipase did not. The relevance of the high affinity complex to the lipase . colipase . substrate complex is discussed.

Animals↗
PubMed1978

Partial purification and properties of monoacylglycerol lipase and two esterases from isolated rat small intestinal epithelial cells.

In intestinal epithelial cells, three enzymes possessing monoacylglycerol hydrolase activity were found and partially purified. Two of these enzymes have properties that justify their classification as an esterase and one as a monoacylglycerol lipase. The three enzymes show similar Km values for monooleoylglycerol and each shows similar activity towards 1- and 2-monopalmitoylglycerol. Antiserum raised in rabbits against rat liver monoacylglycerol lipase inhibits the intestinal lipase completely, suggesting that the enzymes are at least partially similar. The esterases of small intestinal villus cells were not inhibited by the antiserum against liver monoacylglycerol lipase. It was calculated that the esterases account for approx. 2/3 of the monooleoylglycerol hydrolase activity in epithelial cells. Monoacylglycerol lipase also hydrolyzed palmitoyl-CoA, while the esterases did not. The enzymes were inhibited by micellar palmitoyl-CoA. The hypothesis that palmitoyl-CoA is an important regulator for monoacylglycerol acylation is discussed in the light of these new findings.

Acylation↗
PubMed1979

Guinea pig very low density lipoproteins are a good substrate for lipoprotein lipase.

In contrast to plasma from most other animals, guinea pig plasma causes little or no stimulation of lipoprotein lipase activity. Very low density lipoproteins (VLDL) isolated by ultracentrifugation of guinea pig serum caused a definite stimulation of lipase activity, whereas the infranatant inhibited the activity. Gel filtration in 5 M guanidinium hydrochloride of delipidated VLDL demonstrated that the activation was caused by a low molecular weight protein. The VLDL themselves were hydrolized at similar rates as human VLDL both by guinea pig and by bovine lipoprotein lipases. Thus, guinea pig VLDL contain an activator for lipoprotein lipase analogous to that in other animals and there is enough of the activator to support rapid hydrolysis of the VLDL lipids by the lipase.

Animals↗
PubMed1979

Rapid removal to the liver of intravenously injected lipoprotein lipase.

Lipoprotein lipase was purified from bovine milk and labeled with 125I. After intravenous injection to rats the labeled lipase rapidly disappeared from the blood. The initial half-life was about 1 min and more than 70% of the radioactivity was found in the liver at 10 min. 30 min after the injection about 10% of the injected radioactivity was present in acid-soluble form in blood, indicating that the enzyme had been rapidly degraded. Injection of asialofetuin, ribonuclease B or mannan in amounts known to block the hepatic receptors for glycoproteins with exposed galactose, N-acetylglucosamine or mannose residues did not retard the removal of the lipoprotein lipase. Thus, some other, as yet undefined, receptor is implicated. Lipoprotein lipase is known to bind to heparin and some related polysacchrides. Heparin injected before the enzyme delayed its removal and heparin injected after the enzyme caused an immediate increase in blood radioactivity, signifying return from tissues to blood of labeled enzyme. Lipoprotein lipase is present at the endothelium in several extrahepatic tissues and is rapidly turned over. Its presence in blood in appreciable amounts would cause a derangement of lipid transport. The efficient hepatic removal of the enzyme may thus serve an important physiological purpose in keeping the blood levels of this enzyme low.

Animals↗
PubMed1980

Effects of metabolic inhibitors on the synthesis and release of lipoprotein lipase in cultured cells derived from the stromal-vascular fraction of rat adipose tissue.

Cultured cells derived from the stromal-vascular fraction of rat epididymal adipose tissue have been used to study the release of lipoprotein lipase in response to heparin. In the presence of heparin, lipoprotein lipase appeared in the medium biphasically, with an initial rapid rise followed by a slower linear release. The initial increase in activity in the medium was paralleled by a decrease in cell-associated activity. Over the course of the slower phase of release, cell-associated lipoprotein lipase levels remained constant. Treatment of cultures with cycloheximide or 2-deoxyglucose eliminated the second phase of release as well as cell-associated lipoprotein lipase activity. Treatment of cultures with Colcemid decreased the second phase of release but increased cell-associated lipoprotein lipase activity. These data suggest that exposure to heparin results in a rapid release of pre-formed enzyme and that continued release is dependent on both protein synthesis and transport of the enzyme to the cell surface.

Adipose Tissue↗
PubMed1980

Determination of the half-life of lipoprotein lipase in cultured granulosa cells.

The incorporation of [3H]leucine into granulosa lipoprotein lipase was determined by an immunoadsorbent method: a sensitive, specific, and quantitative technique using antiserum to avian adipose lipoprotein lipase coupled to Sepharose 4B as the immunoadsorbent. The sole 3H-labeled protein bound to the immunoadsorbent was shown to be lipoprotein lipase by: (1) sodium dodecyl sulfate polyacrylamide gel electrophoresis, (2) gel filtration on Sephadex G-100, and (3) isoelectric focusing in 8 M urea of the dissociated protein. As measured by a specific radioimmunoassay, all but 4.5% of total enzyme protein, from 1.45 to 380 ng avian lipase, bound to 200 microliters immunoadsorbent. This immunoadsorbent has a quantitative binding capacity (96.5%) over a 200-fold range of enzyme concentration. Using this method, the half-life of lipoprotein lipase under steady-state conditions in cultured granulosa cells was found to be 11.26 h.

Animals↗
PubMed1980

Triacylglycerol metabolism and triacylglycerol lipase activities of cultured human skin fibroblasts.

The relative contribution of lipoproteins and free fatty acid to the cellular triacylglycerol content of cultured human fibroblasts was tested. Fibroblasts accumulated triacylglycerol in proportion to the molar ratio of free fatty acid (oleic acid) to albumin in the medium. Fibroblasts also accumulated triacylglycerol when exposed to medium containing human very low density liproprotein. This accumulation of triacylglycerol was apparently due to direct uptake of intact very low density lipoprotein particles initiated by binding of very low density lipoprotein to cell surface receptors. The amount of 125I-labeled very low density lipoprotein protein internalized and degraded by the cell saturated at the same very low density lipoprotein concentration that produced the maximum increase in cell triacylglycerol. Preincubations with lipoprotein-deficient serum, which enhanced the cell's ability to bind 125I-labeled very low density lipoprotein, increased the amount of 125I-labeled very low density lipoprotein internalized and degraded by the cell in parallel with increased levels of cellular triacylglycerol. Results suggest that the triacylglycerol that accumulates in the presence of very low density lipoprotein represents a lysosomal pool of partially degraded very low density lipoprotein. Measurements of lipase activity of fibroblast homogenates revealed three pH optima at (in descending order of magnitude of activity) pH 4, pH 6, and pH 8. The pH 8 lipase does not appear to represent lipoprotein lipase, since it is not activated by either serum or heparin. Exposure of the cells to medium with varying lipid composition had no effect on the lipase activities. The lipase activities of fibroblasts from donors with familial hypertriglyceridemia appear to be normal.

Adult↗
PubMed1980

Lipoprotein lipase activity in F1 heart cell cultures. Effect of dialyzable serum factors on enzyme stability and enzyme synthesis.

F1 heat cell cultures were grown in F-10 medium containing 20% fetal calf and horse serum and after 6-7 days showed high activity of lipoprotein lipase. When the culture medium contained 20% serum which had been dialyzed against F-10 medium, a 75% decline in lipoprotein lipase activity occurred after 3 h of incubation. Cultures incubated with 20% dialyzed serum and dialysate, obtained after 24 h dialysis of serum against F-10 medium, retained full enzyme activity. Restoration of enzyme activity, lost upon incubation with dialyzed serum, became apparent only 2 h after incubation of the cells with dialysate and dialyzed serum and was complete after 24 h. The effectiveness of the dialysate was not affected by trichloroacetic acid precipitation, ether extraction, exposure to pronase or to 80 degrees C for 10 min; it was retained after chromatography of DEAE cellulose but was lost after elution from CM cellulose colums. Addition of spermidine and spermine to culture media containing dialyzed serum did prevent partially the decline in lipoprotein lipase activity of the heart cell cultures. These polyamines were also able to stabilize lipoprotein lipase activity of heart homogenates incubated at 37 degrees C. However, these compounds were not effective in resoration of enzyme activity of cultured cells lost after exposure to dialyzed serum. It appears that positively charged low molecular weight molecules present in sera of various species are required for the stabilization and synthesis of lipoprotein lipase in heart cell cultures.

Animals↗
PubMed1981

Lipoprotein lipase. Localization on plasma membrane fragments from lactating rat mammary tissue.

Subcellular fractionation studies were done using lactating rat mammary tissue. Lipoprotein lipase (E.C. 3.1.1.34) was found to be associated with plasma membrane-enriched and endoplasmic reticulum-enriched fractions. Considerable amounts of soluble lipoprotein lipase were found after homogenization of this tissue. The membrane-associated lipoprotein lipase activities described here were found to be resistant to release, by heparin, from their membrane sites. Buoyant density gradient fractionation experiments suggest that the lipoprotein lipase activity of lactating mammary tissue is located in part on the secretory epithelial cell plasma membrane. This conclusion is discussed in the context of the known site of action, in vivo, of mammary gland lipoprotein lipase, and of probable routes of its transport to that site.

Animals↗
PubMed1982

Inhibition of lipoprotein lipase by benzene boronic acid. Effect of apolipoprotein C-II.

The catalytic mechanism of triacylglycerol hydrolysis by lipoprotein lipase was studied. We found lipoprotein lipase to be inhibited by benzene boronic acid, with an apparent Ki of 8.9 micro M at pH 7.4. This indicates the presence of serine and histidine in the active site of the enzyme. Inhibition of lipoprotein lipase by benzene boronic acid is likely to be due to the formation of an inhibitor-enzyme complex having analogous bonding to the active site histidine and serine as the transition-state complex which precedes the formation of an obligatory acyl-enzyme intermediate. The presence of apolipoprotein C-II, the apolipoprotein activator of lipoprotein lipase, partly reverses the inhibition of lipoprotein lipase by benzene boronic acid. This reversal by apolipoprotein C-II has a distinct pH optimum in the range of 8-9.

Animals↗