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PubMed1971

Studies on the hormone-sensitive lipase of adipose tissue.

Sucrose gradient centrifugation has been used to examine the triglyceride lipases present in extracts of rat epididymal adipose tissue. The aqueous infranatant recovered between the pellet and fat cake of tissue homogenates which had been centrifuged at 40,000 g was shown to contain two types of triglyceride lipase activity. One of these appears in the 15s region and has been identified as the active form of the "hormone-sensitive lipase" believed to be responsible for initiating the hydrolysis of tissue triglyceride stores in response to lipolytic stimuli. The activity of this enzyme was selectively increased in extracts prepared from tissue exposed to epinephrine and decreased in extracts of insulin-treated tissue. The increased lipolytic activity of extracts of tissue from fasted or fasted-refed rats was also found largely in this region. When the tissue was incubated with orthophosphate-(32)P, radioactivity was incorporated into a protein migrating at 15s. A second peak of triglyceride lipase activity appeared in the 6s region coincident with the location of the monoglyceride and diglyceride lipase activities. The amount of 6s triglyceride lipase activity did not correlate with changes in the lipolytic activity of the tissue from which the extracts were prepared, and its physiological function remains to be elucidated. The lipoprotein lipase and the short-chain triglyceride lipase ("tributyrinase") each moved more slowly in the gradient than the 6s triglyceride lipase. Both the 6s and 15s enzymes were shown to be present in washed adipocytes isolated from the tissue by collagenase digestion.

Adipose Tissue↗
PubMed2001

Contribution of blotting techniques to the study of rapeseeds (Brassica napus L.) lipases.

A recent advance in the study of plant lipases involving immunological techniques is presented. In an attempt to characterize lipases of cotyledons from germinating rapeseed seedlings and to investigate an eventual cross-reactivity with animal lipases, we have prepared anti-porcine pancreatic lipase antibodies raised in rabbit. It is shown by enzyme-linked immunosorbent assay and dot-blotting that these antibodies react with lipases in the rapeseed crude extract and in the different cellular fractions obtained by differential centrifugation. Preincubation of the antiserum with the rapeseed crude extract affects the amount of antibodies binding to the porcine pancreatic lipase. We demonstrate immunochemical cross-reactivity between rapeseed and porcine pancreatic lipase. Using the immunoblotting procedure, it is found that antibodies bind specifically to a single polypeptide with a molecular mass of about 55 kDa. Rapeseed lipase activity decreased after immunoprecipitation suggesting that antibodies were bound to some catalytic site residues. We conclude from the data obtained in this study that the two different lipase species present close similarities in amino acid sequence and antigen characteristics.

Animals↗
PubMed1991

Heterogeneity among ovarian blood vessels: endogenous hepatic lipase is concentrated in blood vessels of rat corpora lutea.

We used indirect immunofluorescence and immunogold light microscopy to examine the distribution of hepatic lipase, an enzyme involved in lipoprotein metabolism, in ovaries of gonadotropin-treated immature rats. Antibodies utilized were rabbit anti-rat hepatic lipase IgG, anti-rat von Willebrand factor (VWF, an endothelial cell marker), and goat anti-rabbit IgG conjugated to gold particles or rhodamine. Immunoreagents were applied to fresh frozen sections of unfixed ovary or liver (positive control) or were delivered to ovaries by vascular perfusion before fixation in situ and silver-enhancement of sections. Appropriate controls verified that the immunolocalizations were specific. Immunofluorescence implied that luteal but not stromal blood vessels of ovaries were positive for hepatic lipase, whereas luteal and stromal blood vessels bore VWF. The improved morphology gained by perfusing ovaries with antibodies allowed precise localization of the enzyme. Hepatic lipase was concentrated within thin-walled vessels of corpora lutea but not those of stroma in ovaries at the time of peak steroidogenic activity. Quantification of hepatic lipase-labeled vessels in stromal and luteal compartments confirmed our visual impression. Many images suggested that stromal vessels lacking hepatic lipase gained this enzyme upon contact with luteal tissue. Perfusion of ovaries with cationized ferritin labeled all ovarian vessels equally well, ruling out the possibility that the observed distribution of hepatic lipase was artifactual. These findings demonstrate that ovarian blood vessels are heterogeneous for hepatic lipase. Moreover, they imply that luteal tissue, perhaps luteal cells, may influence expression of hepatic lipase binding sites by endothelial cells.

Animals↗
PubMed1993

Stereoselective hydrolysis of triglycerides by animal and microbial lipases.

In the present paper, a study on the stereoselectivity of 25 lipases of animal and microbial origin towards homogeneous prochiral triglycerides is presented. All the lipases tested catalyse the hydrolysis of the chemically alike but sterically nonequivalent ester groups in trioctanoin and triolein with different degrees of stereobias, depending on the fatty acyl chain length of the substrate (Rogalska et al., J. Biol. Chem. 256:20271-20276, 1990). Hydrolysis of the sn-2 ester group is catalysed by very few lipases and only Candida antarctica A shows a clear preference for this position. Most of the lipases investigated (12 with trioctanoin and 16 with triolein) showed a preference for the sn-1 position. Using trioctanoin as substrate we observed a total stereoselectivity for position sn-1 with Pseudomonas sp. and Pseudomonas aeruginosa and for position sn-3 with Candida antarctica B. This was not the case with triolein as substrate. Among the 23 lipases studied here and the other two lipases described previously (Rogalska et al., J. Biol. Chem. 256:20271-20276, 1990), 17 show a higher stereoselectivity with trioctanoin than with triolein. With guinea pig pancreatic lipase and with three mold lipases (Geotrichum candidum M, Geotrichum candidum A, and Candida antarctica B), the preference switches from sn-3 to sn-1 when the acyl chain length increases from eight to 18 carbon atoms. The main conclusion to emerge from the present study is that the specific stereopreference of each lipase for a given substrate under given lipolytic conditions can be said to be its fingerprint.

Animals↗
PubMed1999

High-level expression of Rhizopus niveus lipase in the yeast Saccharomyces cerevisiae and structural properties of the expressed enzyme.

Rhizopus niveus lipase (RNL) has a unique structure consisting of two noncovalently bound polypeptides (A-chain and B-chain). To improve this enzyme's properties by protein engineering, we have developed a new expression system for the production of recombinant lipase in the yeast Saccharomyces cerevisiae. For the present study, we developed a more efficient expression system using the strain ND-12B and the multicopy-type plasmid pJDB219. We purified two types of recombinant lipases, each to a single peak by gel-filtration HPLC, although they were found to be heterogeneous by SDS-PAGE. Analysis of reversed-phase HPLC, N-terminal amino acid sequence, and sugar content showed that the difference between the two types of lipases was due mainly to their sugar content (high or low mannose type). Moreover, there were two species within each type of lipase. One kind was processed to the A-chain and B-chain as in the native lipase, while the other remained unprocessed. Although these yeast-purified lipases contained several posttranslational modifications and different glycosylations, their secondary structures were the same as those of the native lipase as measured by circular dichroism spectra and determination of disulfide bonding. This suggests that protein folding of the recombinant lipase occurred correctly in yeast.

Amino Acid Sequence↗
PubMed1987

Influence of iron(III) and pyoverdine on extracellular proteinase and lipase production by Pseudomonas fluorescens B52.

Factors associated with the production of extracellular lipase and proteinase by Pseudomonas fluorescens B52 during the late-log, early-stationary phase of grown were examined. Active lipase production by resting cell suspensions was observed when cells were harvested during the log phase (A600 of 0.3-0.9). Resting suspensions of younger cells (A6000 less than 0.1) synthesized lipase after a significant lag. Addition of cells of the proteinase- and lipase-deficient mutant P. fluorescens RM14 to B52 cells at low density resulted in stimulation of lipase and proteinase production. Similar results were found using cell-free culture fluid of RM14. Gel filtration on Biogel P2 revealed that the stimulatory factor co-chromatographed with the iron(III) siderophore, pyoverdine. Partially purified pyoverdine stimulated enzyme synthesis at a concentration of 6 microM while having no effect on activity of preformed enzyme. Production of pyoverdine and extracellular enzymes was also stimulated by transferrin, a strong iron(III) binding protein. Growth of B52 in deferrated media was limited to 27% of that found with untreated media. Maximum pyoverdine, proteinase and lipase synthesis was obtained at a final iron(III) concentration of 5.75 microM. Growth was maximal in 8.75 microM iron(III) while synthesis of pyoverdine, proteinase and lipase was reduced to 3.6, 6.6 and 30% respectively in 23.75 microM iron(III). Lipase activity in cell-free culture fluid was slightly inhibited by the addition of up to 400 microM iron(III) while proteinase activity was unaffected. In dilute cell suspensions, lipase synthesis was more sensitive to iron(III) than was proteinase (50% inhibition at 1.6 microM and a maximum of 40% inhibition at 5.0 microM, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)

Endopeptidases↗
PubMed1989

Effect of marginal zinc deficiency on lipoprotein lipase activities in postheparin plasma, skeletal muscle and adipose tissues in the rat.

The activities of lipoprotein lipase in postheparin plasma, retroperitoneal adipose and gastrocnemius muscle tissues were determined in the rats fed 2.8 ppm of dietary zinc for eight weeks, as compared with pair-fed and ad libitum-fed rats given 30.8 ppm of zinc. The postheparin lipoprotein lipase activity, as determined by using a lipid emulsion labeled with [3H]triolein as the substrate, was significantly lower in the first group of rats, relative to that in the second and third groups. Tissue lipoprotein lipase activities were compared using the lipid emulsion and activator serum obtained from the zinc-deficient rats and the ad libitum-fed rats. The activator sera were devoid of very low density and low density lipoproteins, but enriched in high density lipoproteins. Muscle lipoprotein lipase activities were significantly lower when assayed with the activator serum from the zinc-deficient compared with the activities determined with the activator serum from the ad libitum-fed. Similarly, muscle lipoprotein lipase activities were lower in all groups when [3H]-triolein-labeled chylomicrons from the zinc-deficient were used as the substrate, compared with the activities determined using the chylomicrons from the ad libitum-fed. Lipoprotein lipase activities in the adipose tissues were not affected by the different sources of the activator sera and chylomicrons. The results strongly suggest that the decrease in postheparin lipoprotein lipase activity in zinc deficiency is not due to changes in tissue lipoprotein lipase enzyme per se, but to compositional alterations in chylomicrons and high density lipoprotein, particularly, with regard to C apolipoproteins, modulators of lipoprotein lipase activity.

Adipose Tissue↗
PubMed2005

Characterization of two novel lipase genes isolated directly from environmental sample.

Two novel lipase genes (lipJ02, lipJ03) were isolated directly from environmental DNA via genome-walking method. Lipase gene lipJ02 contained an open reading frame (ORF) of 1,425 bp and encoded a 474-amino acids lipase protein, while lipase gene lipJ03 contained an ORF of 1,413 bp and encoded a 470-amino acids lipase protein. The lipase genes were cloned into expression vector pPIC9K and successfully integrated into a heterologous fungal host, Pichia pastoris KM71, and the recombinant P. pastoris were screened via a high-throughput method. The recombinants were induced by methanol to secrete active lipases into cultural medium. The recombinant lipases were also purified and characterized. The optimum temperature for the purified lipase LipJ02 and LipJ03 was 30 and 35 degrees C, respectively, at pH 8.0. They exhibited similar thermostability, but LipJ02 exhibited better pH stability than LipJ03.

Chromosome Walking↗
PubMed2004

Purification and characterization of an extracellular lipase from Geotrichum marinum.

An extracellular lipase (EC 3.1.1.3) from Geotrichum marinum was purified 76-fold with 46% recovery using Octyl Sepharose 4 Fast Flow and Bio-Gel A 1.5 m chromatography. The purified enzyme showed a prominent band on SDS-PAGE and a single band on native PAGE based on the activity staining. The molecular mass of the lipase was estimated to be 62 kDa using SDS-PAGE and Bio-Gel A chromatography, indicating that the lipase likely functions as a monomer. The pl of the lipase was determined to be 4.54. The apparent V(max) and Km were 1000 micromol/min/mg protein and 11.5 mM, respectively, using olive oil emulsified with taurocholic acid as substrate. The lipase demonstrated a pH optimum at pH 8.0 and a temperature optimum at 40 degrees C. At 6 mM, Na+, K+, Ca2+, and Mg2+ stimulated activity, but Na+ and K+ at 500 mM and Fe2+ and Mn2+ at 6 mM reduced lipase activity. The anionic surfactant, taurocholic acid, and the zwitterionic surfactant, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, enhanced the activity at 0.1 mM. Other anionic surfactants such as SDS and sodium dioctyl sulfosuccinate, the cationic surfactants methylbenzethonium bromide and cetyltriethylammonium bromide, and the nonionic surfactants Tween-20 and Triton X-100 inhibited the lipase activity to different extents. The lipase was found to have a preference for TG containing cis double bonds in their FA side chains, and the reaction rate increased with an increasing number of double bonds in the side chain. The lipase had a preference for ester bonds at the sn-1 and sn-3 positions over the ester bond at the sn-2 position.

Geotrichum↗
PubMed1999

Lipase-catalyzed fractionation of conjugated linoleic acid isomers.

The abilities of lipases produced by the fungus Geotrichum candidum to selectively fractionate mixtures of conjugated linoleic acid (CLA) isomers during esterification of mixed CLA free fatty acids and during hydrolysis of mixed CLA methyl esters were examined. The enzymes were highly selective for cis-9,trans-11-18:2. A commercial CLA methyl ester preparation, containing at least 12 species representing four positional CLA isomers, was incubated in aqueous solution with either a commercial G. candidum lipase preparation (Amano GC-4) or lipase produced from a cloned high-selectivity G. candidum lipase B gene. In both instances selective hydrolysis of the cis-9,trans-11-18:2 methyl ester occurred, with negligible hydrolysis of other CLA isomers. The content of cis-9, trans-11-18:2 in the resulting free fatty acid fraction was between 94 (lipase B reaction) and 77% (GC-4 reaction). The commercial CLA mixture contained only trace amounts of trans-9,cis-11-18:2, and there was no evidence that this isomer was hydrolyzed by the enzyme. Analogous results were obtained with these enzymes in the esterification in organic solvent of a commercial preparation of CLA free fatty acids containing at least 12 CLA isomers. In this case, G. candidum lipase B generated a methyl ester fraction that contained >98% cis-9,trans-11-18:2. Geotrichum candidum lipases B and GC-4 also demonstrated high selectivity in the esterification of CLA with ethanol, generating ethyl ester fractions containing 96 and 80%, respectively, of the cis-9,trans-11 isomer. In a second set of experiments, CLA synthesized from pure linoleic acid, composed essentially of two isomers, cis-9,trans-11 and trans-10,cis-12, was utilized. This was subjected to esterification with octanol in an aqueous reaction system using Amano GC-4 lipase as catalyst. The resulting ester fraction contained up to 97% of the cis-9,trans-11 isomer. After adjustment of the reaction conditions, a concentration of 85% trans-10,cis-12-18:2 could be obtained in the unreacted free fatty acid fraction. These lipase-catalyzed reactions provide a means for the preparative-scale production of high-purity cis-9,trans-11-18:2, and a corresponding CLA fraction depleted of this isomer.

Chemical Fractionation↗
PubMed1986

Substrate specificities of lipases from corn and other seeds.

Lipases from several seed species were shown to be relatively specific on triacylglycerols containing the major fatty acid components of the storage triacylglycerols in the same species. In a direct comparison using individual triacylglycerol as well as mixed triacylglycerol preparations, highest activities were observed in corn lipase on trilinolein and triolein, castor bean lipase on triricinolein, rapeseed lipase on trierucin, and elm seed lipase on tricaprin. This pattern of fatty acyl specificity was also observed on diacylglycerols, monoacylglycerols, and fatty acyl 4-methylumbelliferone, although the pattern became less distinct. The seed lipases were inactive on lecithins. Corn lipase was more active on tri- than di- or monolinolein, and released linoleic acids from both primary and secondary positions. As judged from the kinetics of hydrolysis of rac-glyceryl-2,3-stearate-1-oleate and rac-glyceryl-1,3-stearate-2-oleate, and of trilinolein and dilinoleins, corn lipase exerted some degree of preference in releasing fatty acid from the primary than the secondary position of a triacylglycerol. At the primary position, corn lipase was more active on oleyl ester than stearyl ester.

Animals↗
PubMed1977

Antibodies to lipoprotein lipase. Application to perfused heart.

An antibody was prepared against purified rat heart lipoprotein lipase. 1. This antibody showed marked species specificity. It inhibited almost totally the lipoprotein lipase activity from all rat tissues examined (i.e., heart, adipose, postheparin plasma, and mammary gland), while having no effect on the activity of lipoprotein lipase partially purified from rabbit, guinea pig and bovine heart and from bovine milk. The antibody also had no effect on the hepatic lipase activity of rat postheparin plasma. 2. After antibody to rat heart lipoprotein lipase was recirculated for 5 min through isolated rat hearts, little or no lipoprotein lipase activity could be detected in the perfusate during 0-20 s of a subsequent non-recirculating perfusion with buffer containing 1 unit heparin/ml. 3. Following recirculation of antibody to lipoprotein lipase for 10 min and a non-recirculating perfusion with buffer for 2 min, the hearts no longer oxidized any significant amounts of 14C-labelled palmitate chylomicron triacylglycerol fatty acid to 14CO2 during a 15-min perfusion. The data give compelling evidence that the functional fraction of lipoprotein lipase in hearts is at the endothelial cell surface accessible to lipoprotein lipase antibody.

Animals↗
PubMed1981

Evidence for the existence of only one triacylglycerol lipase of rat liver active at alkaline pH.

There have been numerous reports suggesting the existence of two or more lipases in liver capable of hydrolyzing triacylglycerols at neutral to alkaline pH. We set out to determine if rat liver contains an alkaline triacylglycerol lipase, in addition to heparin-releasable lipase, which has an intracellular localization. We report here the results of studies concerning the pH dependence, subcellular localization and kinetic analysis of the alkaline lipase(s) of rat liver. Homogenates and cytosolic, microsomal and plasma membrane-enriched subfractions all exhibited an optimum of lipase activity at approx. pH 8.0. In no case was there evidence of multiple pH optima in the alkaline ranges of conformity to Michaelis-Menten kinetics were calculated for the microsomal (0.91 +/- 0.12 mM), cytosolic (1.55 +/- 0.38 mM) and plasma membrane-enriched (1.02 +/- 0.04 mM) subfractions. To determine if the com- and subfractions prepared from control livers with those prepared from livers perfused with collagenase. The loss (93%) of lipase activity from both the cytosolic and microsomal subfractions after collagenase perfusion was identical to the loss (93%) of activity from the homogenates, suggesting a common origin with the collagenase-sensitive alkaline lipase on plasma membrane. The characteristics of hydrolysis in vitro of triacylglycerol contained in artificial and natural substrate preparations by the alkaline lipase of rat liver were examined. The artificial substrate preparation was emulsified tri[1-14C]oleoylglycerol prepared by sonication and the natural substrate preparation was a triacylglycerol-rich lipid fraction ('liver fat') prepared from rat liver homogenates. Although the curves were complex, apparent Km values (mean +/- S.W., n = 3-6) over the limited concentration ranges of conformity to Michaelis-Menten kinetics were calculated for the microsomal (0.91 +/- 0.12 mM), cytosolic (1.55 +/- 0.38 mM) and plasma membrane-enriched (1.02 +/- 0.04 mM) subfractions. To determine if the complexity of these kinetics was related to changes in the products of lipolysis, we examined the products after incubations of plasma membrane-enriched fractions with lower and higher concentrations of triacylglycerol. In either case, the products of lipolysis were diacylglycerol, fatty acids and glycerol; no monoacylglycerol accumulated under any circumstances. At the lower concentrations of either tri[1-14C]oleoylglycerol or liver fat, most triacylglycerol hydrolyzed was degraded fully to fatty acids and glycerol. At the higher triacylglycerol concentrations, while complete degradation continued, virtually all of the increased lipolysis of triacylglycerol (over the lipolysis at the lower substrate concentrations) yielded diacylglycerol. The data indicated that the hydrolysis of diacylglycerol by the alkaline lipase of rat liver occurred at a rate slower than that of triacylglycerol. If the same enzyme catalyzes the lipolysis of both tri- and diacylglycerols, triacylglycerols would appear to be preferred...

Animals↗
PubMed1983

Characteristics of multiple forms of the acidic triacylglycerol lipase(s) of canine cardiac myocytes.

Acidic lipase activity was extracted by digitonin treatment from particulate fractions prepared from isolated adult canine myocytes. Both methylumbelliferyloleate (MUO) and trioleoylglycerol were hydrolyzed with an apparent Km of 13 and 135 microM, respectively. The primary products of trioleoylglycerol lipolysis were oleic acid and 1,2-dioleoylglycerol. Hydrolysis of either MUO or triacylglycerol was stimulated in vitro by the addition of cardiolipin or Triton X-100. Triton X-100 alone was sufficient for maximal stimulation of MUO hydrolysis, but cardiolipin further stimulated triacylglycerol lipolysis in the presence of an optimal concentration of Triton X-100. Cardiolipin increased the Vmax without altering the Km for trioleoylglycerol. Upon gel filtration chromatography the 4-methylumbelliferyloleate and triacylglycerol lipase activities eluted in regions consistent with molecular weights of approx. 47 000 and 55 000, respectively. Chromatofocusing revealed predominantly one form of acidic 4-methylumbelliferyloleate hydrolase (pI approx. 6.3), whereas acidic triacylglycerol lipase activity eluted continuously in the pH gradient from 7.2 to 4.3 with no clearly predominant peak of activity. Two forms of both 4-methylumbelliferyloleate and triacylglycerol lipase were eluted from columns of carboxymethyl Bio-Gel at pH 5.7; one form of each lipase activity was not bound and another form of each lipase was eluted with 50-60 mM KCl. The non-bound forms of each lipase were indistinguishable from their respective carboxymethyl-bound forms on the basis of pH dependency or kinetically (similar Km). The non-bound and carboxymethyl-bound peaks of lipolytic activity differed in the ratios of 4-methylumbelliferyloleate hydrolase to triacylglycerol lipase activity. The results suggest that the cardiac myocyte contains multiple forms of acidic lipase, and that the catalytic units primarily responsible for the hydrolysis of methylumbelliferyl esters and triacylglycerols may not be identical.

Animals↗
PubMed1983

Transfer of cholesteryl linoleyl ether from phosphatidylcholine and phosphatidylethanolamine liposomes to cultured cells catalyzed by lipoprotein lipase.

Unilamellar liposomes prepared from purified phospholipids (phosphatidylcholine, phosphatidylethanolamine or sphingomyelin) and labeled cholesteryl linoleyl ether were used to study lipoprotein lipase-catalyzed transfer of cholesteryl ester into cells in culture. In mesenchymal rat heart cell cultures, the transfer of cholesteryl linoleyl ether and cholesteryl linoleate was similar and related to the activity of endogenously produced lipoprotein lipase. In human skin fibroblasts transfer of labeled cholesteryl linoleyl ether was proportional to the concentration of milk lipoprotein lipase added to the incubation medium. Liposomes prepared from phosphatidylcholine or phosphatidylethanolamine were much better donors of cholesteryl ether to normal and apolipoprotein E-B receptor-negative fibroblasts and to endothelial cells than those prepared from sphingomyelin. Lysophosphatidylcholine was formed during incubation with milk lipoprotein lipase but was not considered to be directly responsible for the lipoprotein lipase-catalyzed transfer of cholesteryl ether. This conclusion was drawn because in the absence of lipoprotein lipase addition of lysophosphatidylcholine to liposomes, or almost complete phospholipolysis by phospholipase A2, did not result in the transfer of cholesteryl linoleyl ether from liposomes to cells. Attachment of lipoprotein lipase to the cell surface was mandatory for the transfer of cholesteryl ether and could be prevented by heparin. High density apolipoprotein reduced also the transfer of cholesteryl linoleyl ether, even though it did not interfere with the binding of labeled milk lipoprotein lipase to cultured fibroblasts. The present results provide evidence that lipoprotein lipase, and not the products of phospholipid hydrolysis, is the ligand for the non-apolipoprotein E-B receptor-mediated transfer of cholesteryl ester to cells.

Animals↗
PubMed1983

Influence of nutritional state on lipoprotein lipase activities in the hypothyroid rat.

We have investigated the effects of nutritional state on the lipoprotein lipase activities of the experimentally hypothyroid rat. Both short-term effects (i.e., those of a 24 h fast with and without re-feeding) and long-term effects (due to decreased food intake in hypothyroidism) have been studied. The hypothyroid rats had significantly higher lipoprotein lipase activities of adipose tissue and heart muscle. The effect of hypothyroidism on adipose tissue lipoprotein lipase activities was modified by the nutritional state. In rats studied after 24 h fasting, the hypothyroid group had significantly higher lipoprotein lipase activities than weight-matched, age-matched and pair-fed (i.e., semi-starved) control groups. In rats studied in the re-fed state, the effects of hypothyroidism as such were less evident, since the pair-fed group also demonstrated significantly higher enzyme activities than did the other control groups. We have also studied the lipoprotein lipase activities of different enzyme preparations from adipose tissue. The effects of hypothyroidism were most clearly reflected in an increase of heparin-elutable enzyme activity from adipose tissue, whereas adipocyte lipoprotein lipase activity and the lipoprotein lipase secretion rate from adipocytes were affected to a lesser extent. We conclude that alterations in food intake strongly influence the lipoprotein lipase activities in the hypothyroidism. Our data also imply that the increased lipoprotein lipase activity in the hypothyroid state is due to a decreased degradation of the enzyme, both intra- and extracellularly.

Adipose Tissue↗
PubMed1985

Subcellular localization and properties of lipase activities in human polymorphonuclear leukocytes.

A fluorimetric assay for lipase activity has been optimized for measurement of the enzyme in human neutrophils. Activity was maximal at acid (4.5) and alkaline (9.5) pH, although there was also a neutral peak of activity at pH 6.5. Neutrophils were homogenised in isotonic sucrose and subjected to analytical subcellular fractionation by sucrose density gradient centrifugation. The gradient fractions were assayed for acid, neutral and alkaline lipase activity and for the principal organelle marker enzymes. Neutral lipase showed a unimodal distribution with an equilibrium density of 1.19 g . cm-3, corresponding to the distribution of particulate leucine aminopeptidase. Acid and alkaline lipase activities showed very similar distribution profiles to each other with both soluble components and a broad peak of particulate activity. The broad modal density of 1.19-1.22 g . cm-3 suggests that acid and alkaline lipase activities could be localised to more than one population of cytoplasmic granule. Fractionation experiments with neutrophils homogenised in sucrose medium containing digitonin confirmed the localisation of neutral lipase and leucine aminopeptidase to the same cytoplasmic granule, and suggested that at least part of the acid lipase activity was localised to the specific granule. No lipase activity could be attributed to the alkaline phosphatase-containing granule. Neutrophils were isolated from control subjects, patients with chronic granulocytic leukaemia and women in the third trimester of pregnancy. The specific activity of acid, neutral and alkaline lipase, and leucine aminopeptidase, in contrast to that of alkaline phosphatase, were similar in the three patient groups.

Adult↗
PubMed1985

Antibody against rat adipose tissue lipoprotein lipase.

To facilitate detailed studies of rat adipose tissue lipoprotein lipase regulation, a high titre polyclonal antibody was raised against purified rat adipose tissue lipoprotein lipase (in a goat). The first stage of the purification of the lipoprotein lipase was carried out with heparin-Sepharose affinity chromatography. In the second stage we took advantage of the binding property of lipoprotein lipase to ampholytes. These ampholytes, used during this second step, do not have to be eliminated prior to injecting the enzyme preparation into the animal. They have neither toxic nor antigenic effects on the animal; moreover, their presence does not affect the antigenic potency of the lipoprotein lipase. When pre-incubated with a constant amount of adipose tissue lipoprotein lipase (8 mU/75 microliter), an equal volume of the antiserum raised either pure or diluted up to 1/50 resulted in complete inhibition of enzyme activity, and half maximal inhibition was observed at a dilution of 1/800. The antibody was effective in inhibiting rat heart lipoprotein lipase but not salt-resistant hepatic lipase. Immunodiffusion revealed a single line of precipitation between this antibody and the adipose tissue lipoprotein lipase.

Adipose Tissue↗