The lipoprotein lipase of white adipose tissue. Studies on the intracellular distribution of the adipocyte-associated enzyme.

The separation of rat epididymal adipocytes into plasma-membrane, mitochondrial, microsomal and cytosol fractions is described. The fractions, which were characterized by marker-enzyme analysis and electron-micrographic observation, from the cells of fed and 24 h-starved animals were used to prepare acetone/diethyl ether-dried powders for the measurement of lipoprotein lipase activities. The highest specific activities and proportion of recovered lipoprotein lipase activity were found in the plasma-membrane and microsomal fractions. The two fractions from the cells of fed rats showed similar activities and enrichments of the enzyme, these activities being higher than the plasma-membrane and lower than the microsomal activities recovered from the cells of starved animals. Chicken and guinea-pig anti-(rat lipoprotein lipase) sera were prepared, and an indirect labelled-second-antibody cellular immunoassay, using 125I-labelled rabbit anti-(chicken IgG) or 125I-labelled sheep anti-(guinea-pig IgG) antibodies respectively, for the detection of cell-surface enzyme was devised and optimized. The amount of immunodetectable cell-surface lipoprotein lipase was higher for cells isolated from fed animals than for cells from 24 h-starved animals, when either anti-(lipoprotein lipase) serum was used in the assay. The amount of immunodetectable cell-surface lipoprotein lipase fell further when starvation was extended to 48 h. The lipoprotein lipase of plasma-membrane vesicles was shown to be a patent activity and to be immunodetectable in a modification of the cellular immunoassay. Although the functional significance of the adipocyte surface lipoprotein lipase is not known, the possibility of it forming a pool of enzyme en route to the capillary endothelium is advanced.     

Studies on the effect of bile salt and colipase on enzymatic lipolysis. Improved method for the determination of pancreatic lipase and colipase.

The rate of hydrolysis of long chain triglycerides by pure bovine pancreatic lipase has been determined in the presence of variable amounts of bile salts and colipase. Cofactor-free lipase is strongly inhibited by sodium taurodesoxycholate and by mixed bovine bile salts at concentrations higher than the critical micellar concentration. Bile salt inhibited lipase is reactivated by the addition of bovine colipase. Gel filtration of pancreatic juice from several species (Cow, dog, pig) on Sephadex G 100 allows the separation of lipase from colipase. It is found that the enzyme catalyzed hydrolysis of long chain triglycerides by pancreatic lipase from one species is activated by the addition of colipase from other species. Studies on the activation of pancreatic lipase by colipase in the presence of bile salts allowed the re-evaluation of optimal conditions for the determination of lipase and the development of a procedure to assay colipase.     

Regulation of lipoprotein lipase. Induction by insulin.

Lipoprotein lipase activity in intact epididymal adipose tissue of fasted rats increased rapidly after treatment with insulin in vivo. In contrast, lipoprotein lipase activity in adipocytes isolated from the contralateral fat pads remained essentially unchanged. When adipocytes were incubated for 30 min at ambient temperature in vitro, about 2 times more lipoprotein lipase activity was found in the medium of cells from insulin-treated rats than in medium from cells of control animals. Following insulin treatment, extracts of tissue acetone powders separated by gel chromatography showed increases in both enzyme activity fractions obtained (designated lipoprotein lipase a and b). However, no consistent differences were observed between fractions derived from adipocyte acetone powders of insulin-treated and control animals. All the observed effects of insulin on lipoprotein lipase activity were abolished by cycloheximide treatment in vivo. These data indicate that following insulin treatment, increased lipoprotein lipase activity in adipose tissue results from enhanced enzyme secretion by the fat cell and subsequent accumulation in the tissue, thus implicating the adipocyte secretory mechanism as a major site of regulation of lipoprotein lipase activity in adipose tissue.     

Purification and properties of the extracellular lipase, LipA, of Acinetobacter sp. RAG-1.

An extracellular lipase, LipA, extracted from Acinetobacter sp. RAG-1 grown on hexadecane was purified and properties of the enzyme investigated. The enzyme is released into the growth medium during the transition to stationary phase. The lipase was harvested from cells grown to stationary phase, and purified with 22% yield and > 10-fold purification. The protein demonstrates little affinity for anion exchange resins, with contaminating proteins removed by passing crude supernatants over a Mono Q column. The lipase was bound to a butyl Sepharose column and eluted in a Triton X-100 gradient. The molecular mass (33 kDa) was determined employing SDS/PAGE. LipA was found to be stable at pH 5.8-9.0, with optimal activity at 9.0. The lipase remained active at temperatures up to 70 degrees C, with maximal activity observed at 55 degrees C. LipA is active against a wide range of fatty acid esters of p-nitrophenyl, but preferentially attacks medium length acyl chains (C6, C8). The enzyme demonstrates hydrolytic activity in emulsions of both medium and long chain triglycerides, as demonstrated by zymogram analysis. RAG-1 lipase is stabilized by Ca2+, with no loss in activity observed in preparations containing the cation, compared to a 70% loss over 30 h without Ca2+. The lipase is strongly inhibited by EDTA, Hg2+, and Cu2+, but shows no loss in activity after incubation with other metals or inhibitors examined in this study. The protein retains more than 75% of its initial activity after exposure to organic solvents, but is rapidly deactivated by pyridine. RAG-1 lipase offers potential for use as a biocatalyst.     

Cloning of the Pseudomonas glumae lipase gene and determination of the active site residues.

The lipA gene encoding the extracellular lipase produced by Pseudomonas glumae PG1 was cloned and characterized. A sequence analysis revealed an open reading frame of 358 codons encoding the mature lipase (319 amino acids) preceded by a rather long signal sequence of 39 amino acids. As a first step in structure-function analysis, we determined the Ser-Asp-His triad which makes up the catalytic site of this lipase. On the basis of primary sequence homology with other known Pseudomonas lipases, a number of putative active site residues located in conserved areas were found. To determine the residues actually involved in catalysis, we constructed a number of substitution mutants for conserved Ser, Asp, and His residues. These mutant lipases were produced by using P. glumae PG3, from which the wild-type lipase gene was deleted by gene replacement. By following this approach, we showed that Ser-87, Asp-241, and His-285 make up the catalytic triad of the P. glumae lipase. This knowledge, together with information on the catalytic mechanism and on the three-dimensional structure, should facilitate the selection of specific modifications for tailoring this lipase for specific industrial applications.     

Cloning of the Pseudomonas glumae lipase gene and determination of the active site residues.

The lipA gene encoding the extracellular lipase produced by Pseudomonas glumae PG1 was cloned and characterized. A sequence analysis revealed an open reading frame of 358 codons encoding the mature lipase (319 amino acids) preceded by a rather long signal sequence of 39 amino acids. As a first step in structure-function analysis, we determined the Ser-Asp-His triad which makes up the catalytic site of this lipase. On the basis of primary sequence homology with other known Pseudomonas lipases, a number of putative active site residues located in conserved areas were found. To determine the residues actually involved in catalysis, we constructed a number of substitution mutants for conserved Ser, Asp, and His residues. These mutant lipases were produced by using P. glumae PG3, from which the wild-type lipase gene was deleted by gene replacement. By following this approach, we showed that Ser-87, Asp-241, and His-285 make up the catalytic triad of the P. glumae lipase. This knowledge, together with information on the catalytic mechanism and on the three-dimensional structure, should facilitate the selection of specific modifications for tailoring this lipase for specific industrial applications.     

Fate of lipoprotein lipase taken up by the rat liver. Evidence for a conformational change with loss of catalytic activity

When isolated rat livers were perfused with medium containing lipoprotein lipase, 40-60% was taken up during a single passage. This value was similar for lipoprotein lipase derived from culture medium of rat preadipocytes, and for lipoprotein lipase purified from bovine milk. It was also, similar, irrespective of the lipoprotein lipase concentration, at least up to 1 microgram/ml. Immediately following its uptake by the liver, a large fraction of the lipoprotein lipase could be released by heparin, but the magnitude of this fraction decreased with time. The enzyme lost its catalytic activity rather rapidly, but its degradation to acid-soluble products, or to larger fragments, was much slower. On heparin-agarose chromatography, the enzyme taken up by the liver eluted at a lower salt concentration than the original lipoprotein lipase preparation. This change in affinity for heparin suggests that the originally dimeric lipoprotein lipase had dissociated into monomers, in analogy to the findings in model experiments. It is suggested that the initial uptake of lipoprotein lipase occurs by binding to a polyanion at the liver cell surface. This is followed by endocytosis and dissociation of the enzyme from its heparan sulfate-like binding site. Acidification of the endosome may cause a conformational change in the lipase molecule with dissociation to inactive monomers, preceding ultimate proteolytic degradation.     

Penicillium sp.脂肪酶的发酵及催化生成生物柴油的研究

目的：为了提高脂肪酶的产量及更好地应用脂肪酶。方法：采用单因子实验与均匀设计相结合的方法,对青霉Penicil- lium sp．TS414发酵生产脂肪酶的条件进行了优化。结果：在实验优化后的最适产酶培养基中,碳源为1．4%蔗糖,氮源为7．0%豆饼粉,起始pH8．0。均匀设计优化后的产酶水平(315．1U/mL)比优化前(101．5U/mL)提高了约2倍。Penicillium sp．TS414脂肪酶能够有效地催化大豆油转酯化合成脂肪酸甲酯(生物柴油),反应72h后,脂肪酸甲酯的最终得率在96%左右。结论：Penicillium sp．TS414产生的脂肪酶在生物柴油的工业化生产方面,具有潜在的应用前景。     

Hormone-sensitive lipase of adipose tissue.

Some physiologic aspects of the mobilization and fate of free fatty acids are reviewed. The molecular mechanism of the activation of hormone-sensitive lipase in adipose tissue is then discussed. Recent evidence established that hormone-sensitive lipase, concerned with fat mobilization, is both functionally and immunochemically distinct from lipoprotein lipase, concerned with uptake of plasma triglycerides. Lipoprotein lipase activity is not altered by cyclic AMP-dependent protein kinase. The latter enzyme enhances not only triglyceride hydrolase but also monoglyceride, diglyceride and cholesterol ester hydrolase activities in chicken adipose tissue. Finally, it is shown that the activation of all four acyl hydrolases is reversible, the deactivation being magnesium-dependent. Protein phosphatase fractions from heart and liver active against phosphorylase a can reversibly deactivate adipose tissue hormone-sensitive lipase, implying a low degree of substrate specificity for lipase phosphatase.     

Purification and characterization of a lipase from the glycolipid-producing yeast Kurtzmanomyces sp. I-11

An extracellular lipase produced by the glycolipid-producing yeast Kurtzmanomyces sp. I-11 was purified by ammonium sulfate precipitation and column chromatographies on DEAE-Sephadex A-25, SP-Sephadex C-50, and Sephadex G-100. Based on the analysis of the purified lipase on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the purified lipase was judged to be homogeneous and its molecular mass was estimated to be approximately 49 kDa. The optimum temperature for the activity was 75 degrees C, and the activity was very stable at temperatures below 70 degrees C. The active pH range of this lipase was 1.9-7.2, and the activity was stable at pH below 7.1. The lipase showed a preference for C18 acyl groups by measurements with p-nitrophenyl esters and triglycerides as substrates. The lipase was very stable in the presence of various organic solvents at a concentration of 40%. Although the N-terminal sequence of the Kurtzmanomyces lipase was very similar to that of lipase A from Candida antarctica, the pH profiles of the two lipases were significantly different.     

Inhibition of pancreatic lipase in vitro by the covalent inhibitor tetrahydrolipstatin.

Tetrahydrolipstatin inhibits pancreatic lipase from several species, including man, with comparable potency. The lipase is progressively inactivated through the formation of a long-lived covalent intermediate, probably with a 1:1 stoichiometry. The lipase substrate triolein and also a boronic acid derivative, which is presumed to be a transition-state-form inhibitor, retard the rate of inactivation. Therefore, in all probability, tetrahydrolipstatin reacts with pancreatic lipase at, or near, the substrate binding or active site. Tetrahydrolipstatin is a selective inhibitor of lipase; other hydrolases tested were at least a thousand times less potently inhibited.     

Developmental regulation of hormone-sensitive lipase mRNA in the rat: changes in steroidogenic tissues.

The hydrolysis of triglycerides and cholesteryl esters stored within cells is mediated by the enzyme, hormone-sensitive lipase. In adipose tissue and heart, hormone-sensitive lipase primarily hydrolyzes stored triglycerides to free fatty acids, while in steroidogenic tissues, it principally converts cholesteryl esters to free cholesterol for steroid hormone production. To determine whether hormone-sensitive lipase is under tissue-specific, developmental regulation, the steady state levels of hormone-sensitive lipase mRNA were determined in normal rats from late fetal life through 2 years of age. Hormone-sensitive lipase mRNA levels did not appear to vary in adipose tissue from epididymal fat pads obtained from animals between 3 weeks and 2 years of age. In heart, hormone-sensitive lipase mRNA levels were lowest in the fetus increased rapidly within the first day postnatally, and then gradually increased to stable adult levels by 2 months that were 3-fold higher than observed in fetal rats. Steady state mRNA levels of hormone-sensitive lipase in the adrenals were lowest in fetal rats, increased 4-fold during the first day and peaked at levels that were 9-fold higher by the end of the first week. Thereafter, levels fell and remained 3- to 4-fold higher than at birth throughout adult life. Hormone-sensitive lipase mRNA was undetectable in testes before 4 weeks of age and increased 25-fold to stable adult levels between 4 and 12 weeks. Thus, hormone-sensitive lipase is differentially expressed and regulated in a tissue-specific fashion during development and aging.     

The effect of lipase from Penicillium sp. on the membrane fraction of the levorin-producing Streptomyces levoris

The effect of a lipase preparation from Penicillium sp. on the membranes of the levorin producer Streptomyces levoris was being studied. The enzyme preparation was found preferably to hydrolyse neutral lipids in the Str. levoris membranes, which makes it possible to use the lipase from Penicillium sp. for studying neutral lipids in microbial membranes.     

On the interactions between pancreatic lipase and colipase and the substrate, and the importance of bile salts.

The interactions between pancreatic lipase and colipase and the substrate and the effect of bile salts on these interactions have been investigated by the use of kinetic experiments and studies on the semiquantitative phase distribution of lipase and colipase activities. The results suggest that lipase binds to hydrophobic interfaces with partial irreversible inactivation. Bile salts in the range of micellar concentrations and above a pH of about 6.5 displace lipase from this binding, resulting in a reversible in activation. At pH values below about 6.5, lipase binds strongly to the substrate even in the presence of bile salt, and a low activity peak is seen around pH 5.5. This is the result of the binding of lipase to the "supersubstrate" and the activity of the catalytic site. In the presence of bile salt, colipase promotes the binding of lipase to the "supersubstrate" but not to other hydrophobic interfaces, and catalytic activity is reestablished. Kinetic data indicate that the binding between colipase and lipase in the presence of substrate is strong and occurs in an approximately stoichiometric relationship.     

Effect of dimethylsulfoxide on hydrolysis of lipase.

To establish an industrially feasible reaction process, the effect of dimethylsulfoxide (DMSO) added to an aqueous solution on the hydrolysis of lipase was investigated using fluorescent substrates. Several lipases from microorganisms were improved in their hydrolysis activities against 4-methylumbelliferyl oleate by DMSO. Variation was found in the effect of DMSO depending on the species of lipase. After the high stability of the lipase from Pseudomonas fluorescens in DMSO solution was confirmed, hydrolysis by this lipase of four acyl-4-methylumbelliferones was studied kinetically at different DMSO concentrations. DMSO added to an aqueous solution increased the Vmax of this lipase for a substrate with strong hydrophobicity, and decreased that value for a substrate with an opposite property. On the other hand, DMSO had a very small effect on Km for each substrate. A fluorometric study suggested that DMSO induced a change of the chemical environment that surrounded tryptophan residues of the lipase. Such conformational change would be one of the causes of the DMSO-induced alteration of its reactive property. These results suggest that the addition of DMSO may be a novel method of 'solvent engineering' of this enzyme.     

Maturation and secretion of lipoprotein lipase in cultured adipose cells. I. Intracellular activation of the enzyme

The intracellular pathway and the activation of lipoprotein lipase have been examined in differentiated Ob17 cells. These adipose cells were previously shown to secrete lipoprotein lipase during exposure to heparin. Treatment of the cells with cycloheximide and heparin leads to enzyme depletion, as shown by activity measurement and immunofluorescence microscopy. The repletion phase has been studied in the presence of monensin or carbonyl cyanide m-chlorophenylhydrazone, ionophores known to affect the intracellular transport of membrane and secretory proteins. Monensin-treated cells synthesize fully active lipoprotein lipase. Under these conditions the antigen accumulates in the Golgi apparatus and the heparin-stimulated enzyme release is extensively reduced. Carbonyl cyanide m-chlorophenylhydrazone-treated cells do not contain any enzyme activity but show detectable antigen which accumulates in the endoplasmic reticulum. Competition for binding to immobilized anti-lipoprotein lipase antibodies of mature and endoplasmic reticulum-sequestered antigens is observed. Carbonyl cyanide m-chlorophenylhydrazone removal is rapidly followed by a transient burst of enzyme activity and a redistribution of the antigen in the different subcellular compartments. Therefore, the results show that the activation of lipoprotein lipase is an intracellular event taking place after the enzyme exits from the endoplasmic reticulum and before it reaches the trans-Golgi cisternae.     

Identification of the active-site serine in human pancreatic lipase by chemical modification with tetrahydrolipstatin.

A chemical modification approach was used in this study to identify the active site serine residue of human pancreatic lipase. Purified human pancreatic lipase was covalently modified by incubation with [3H], [14C] tetrahydrolipstatin (THL), a potent inhibitor of pancreatic lipase. The radiolabeled lipase was digested with thermolysin, and the peptides were separated by HPLC. A single THL-peptide-adduct was obtained which was identical to that obtained earlier from porcine pancreatic lipase. This pentapeptide with the sequence VIGHS is covalently bound to a THL molecule via the side chain hydroxyl group of the serine unit corresponding to Ser-152 of the lipase. The selective cleavage of the THL-serine bond by mild acid treatment resulted in the formation of the delta-lactone Ro 40-4441 in high yield and clearly proves that THL is attached via an ester bond and with retention of stereochemistry at all chiral centers to the side chain hydroxyl group of Ser-152 of the lipase. The results obtained for human pancreatic lipase corroborate the inhibition mechanism of THL found on the porcine enzyme, and are in full agreement with the identification of the Ser-152 ... His-263 ... Asp-176 catalytic triad in the X-ray structure of human pancreatic lipase.     

Lipoprotein lipase activity of rat cardiac muscle. The intracellular distribution of the enzyme between fractions prepared from cardiac muscle and cells isolated from the hearts of fed and starved animals.

1. Subcellular fractions, characterized by using morphological, compositional and enzymic markers, were prepared from rat heart tissue and cells isolated from the hearts of fed and 24 h-starved rats. 2. The lipoprotein lipase activity of fractions from whole tissue and isolated cells was determined in either fresh fractions or in acetone/diethyl ether powders of the fractions. 3. Lipoprotein lipase activity was present in all the fractions from tissue and cells, but was found to be of highest relative specific activity in the microsomal () fractions. 4. In fractions prepared from the isolated cells of hearts from starved rats the proportion of the total lipoprotein lipase present and its relative specific activity in the microsomal fraction were greater than in the equivalent fractions from fed animals. 5. The enhancement of lipoprotein lipase activity as a result of the acetone/diethyl ether powder preparation of fractions was most extensive in the microsomal fractions. 6. Investigation of the microsomal fraction showed that the lipoprotein lipase activity present was in two pools, one of which was within endoplasmic-reticulum vesicles. 7. The observations were consistent with the possibility that the cardiac-muscle cell could be the origin of the lipoprotein lipase activity functional in triacylglycerol uptake by the heart.     

The lipoprotein lipase of white adipose tissue. Changes in the adipocyte cell-surface content of enzyme in response to extracellular effectors in vitro.

An indirect labelled-second-antibody cellular immunoassay for adipocyte surface lipoprotein lipase was used to assess the changes that occurred during the incubation of cells in the presence and absence of effectors. In the absence of any specific effectors, the amount of immunodetectable lipoprotein lipase present at the surface of adipocytes remained constant throughout the 4 h incubation period at 37 degrees C. Under such conditions total cellular enzyme activity also remained constant, with no activity appearing in the medium. In the presence of heparin, cell-surface immunodetectable lipoprotein lipase increased by up to 20%, whereas in the presence of cycloheximide they decreased by up to 60%. Thus the obvious turnover of enzyme from this cell-surface site was found to be relatively rapid and dependent for its replenishment, at least in part, on protein synthesis. In the presence of insulin alone, a substantial increase in cell-surface lipoprotein lipase protein occurred, only part of which was dependent on protein synthesis. The total cellular activity of lipoprotein lipase was unaffected by the presence of insulin. The insulin-dependent increase in cell-surface enzyme was potentiated somewhat in the presence of dexamethasone, which was not shown to exert any independent effect. Glucagon, adrenaline and theophylline all produced a significant decline in the cell-surface immunodetectable lipoprotein lipase, which in the case examined (adrenaline) was partially additive with regard to the independent effect of cycloheximide. Cell-surface immunodetectable lipoprotein lipase amounts were decreased significantly when cells were incubated in the presence of either colchicine or tunicamycin. The concerted way in which cell-surface lipoprotein lipase altered during the incubations of adipocytes in the presence of effectors suggested that the translocation of enzyme to and from this cellular site was dependent on hormonal action and the integrity of intracellular protein-transport mechanisms.     

Stereoselectivity of lipases. II. Stereoselective hydrolysis of triglycerides by gastric and pancreatic lipases

In the present study, porcine pancreatic lipase, rabbit gastric lipase, and human gastric lipase stereospecificity toward chemically alike, but sterically nonequivalent ester groups within one single triglyceride molecule was investigated. Lipolysis reactions were carried out on synthetic trioctanoin or triolein, which are homogenous, prochiral triglycerides, chosen as models for physiological lipase substrates. Diglyceride mixtures resulting from lipolysis were derivatized with optically active R-(+)-1-phenylethylisocyanate, to give diastereomeric carbamate mixtures, which were further separated by high performance liquid chromatography. Resolution of diastereomeric carbamates gave enantiomeric excess values, which reflect the lipases stereobias and clearly demonstrate the existence of a stereopreference by both gastric lipases for the sn-3 position. The stereoselectivity of human and rabbit gastric lipases, expressed as the enantiomeric excess percentage, was 54% and 70% for trioctanoin and 74% and 47% for triolein, respectively. The corresponding values with porcine pancreatic lipase were 3% in the case of trioctanoin and 8% in that of triolein. It is worth noting that rabbit gastric lipase, unlike human gastric lipase, became more stereoselective for the triglyceride with shorter acyl chains (trioctanoin). This is one of the most striking catalytic differences observed between these two gastric lipases.