Reversible protein kinase activation of hormone-sensitive lipase from chicken adipose tissue

Hormone-sensitive lipase partially purified from adipose tissue of laying hens was markedly activated by cyclic AMP-dependent protein kinase. Activation was approximately 4-fold (ranging up to as great as 10-fold) compared with the much lower degree of activation obtained with analogous preparations from rat and human adipose tissues (59 and 86%, respectively). The partially purified preparations contained adequate endogenous protein kinase activity to effect complete activation with addition of cyclic AMP, ATP, and Mg(2+). Activation was blocked by protein kinase inhibitor (from rabbit skeletal muscle) but could be restored fully by addition of excess exogenous protein kinase (from bovine skeletal muscle). The fully activated lipase was slowly deactivated by dialysis at 4 degrees C and then rapidly and almost fully reactivated by addition of cyclic AMP and ATP-Mg(2+). Reactivation was blocked by protein kinase inhibitor. This deactivation-reactivation cycle was rapid at 23 degrees C with dialysis against charcoal and could be demonstrated repeatedly using a single preparation. The reversible deactivation of protein kinase-activated enzyme is presumed to reflect the action of a lipase phosphatase. Lipase prepared from tissue previously exposed to glucagon yielded a much smaller degree of activation than lipase prepared from tissue not exposed to the lipolytic hormone, indicating that the physiological hormone-induced activation is probably similar to or identical with the protein kinase activation demonstrated in the cell-free preparations. Under the conditions of assay used, the partially purified lipase fraction contained diglyceride, monoglyceride, and lipoprotein lipase activities. However, treatment with cyclic AMP-dependent protein kinase had virtually no effect on these lipase activities.     

Intracellular lipase activities in heart and skeletal muscle homogenates. The absence of trierucin cleavage by the heart: a possible biochemical basis for erucic acid lipidosis.

Rat heart and skeletal muscle homogenates were compared for their intracellular lipolytic activity towards a series of saturated and unsaturated triglycerides from trilaurin (C12:0) to trierucin (C22:1). It is shown that for all triglycerides esterified with fatty acids from C12 to C18, lipolytic activity in heart homogenates was higher than in skeletal muscle homogenates. For these triglycerides there was no relationship between the fatty acid chain length and the lipolytic activity. In both homogenates cleavage of unsaturated triglycerides was higher than cleavage of the homologous saturated triglyceride. Lipolysis of tri-delta-11-eicosenoin (C20:1) was similar in both homogenates but much lower than lypolysis of other triglycerides. Although cleavage of trierucin (C22:1) was very low in skeletal muscle homogenates, it was undetectable in heart homogenates, even when enzyme concentration was increased. A mixture of triglycerides did not show preferential hydrolysis of any simple triglyceride. Trierucin was the only triglyceride that did not complete for lipolytic activity and only with heart homogenates, which shows that that lipase(s) do not cleave trierucin. The absence of lipolytic activity towards trierucin in heart homogenates could explain the selective accumulation of erucic acid-rich triglycerides in hearts of animals fed a diet with a high erucic acid content.     

Cloning and sequencing of the triacylglycerol lipase gene of Aspergillus oryzae and its expression in Escherichia coli

Aspergillus oryzae produces at least three extracellular lipolytic enzymes, L1, L2 and L3 (cutinase, mono- and diacylglycerol lipase, and triacylglycerol lipase, respectively). We cloned the triacylglycerol lipase gene (provisionally designated tglA) by screening a genomic library using a PCR product obtained with two degenerate oligonucleotide primers corresponding to amino acid sequences of L3 as probes. Nucleotide sequencing of the genomic DNA and cDNA revealed that the L3 gene (tglA) has an open reading frame comprising 954 nucleotides, which contains three introns of 47, 83 and 62 bp. The deduced amino acid sequence of the tglA gene corresponds to 254 amino acid residues including a signal sequence of 30 amino acids and, in spite of the difference in substrate specificity, it is homologous to those of cutinases from fungi. Three residues presumed to form the catalytic triad, Ser, Asp and His, are conserved. The cloned cDNA of the tglA gene was expressed in Escherichia coli, and enzyme assaying and zymography revealed that the cloned cDNA encodes a functional triacylglycerol lipase.     

Cellular Localization and Associations of the Major Lipolytic Proteins in Human Skeletal Muscle at Rest and during Exercise

Lipolysis involves the sequential breakdown of fatty acids from triacylglycerol and is increased during energy stress such as exercise. Adipose triglyceride lipase (ATGL) is a key regulator of skeletal muscle lipolysis and perilipin (PLIN) 5 is postulated to be an important regulator of ATGL action of muscle lipolysis. Hence, we hypothesized that non-genomic regulation such as cellular localization and the interaction of these key proteins modulate muscle lipolysis during exercise. PLIN5, ATGL and CGI-58 were highly (>60%) colocated with Oil Red O (ORO) stained lipid droplets. PLIN5 was significantly colocated with ATGL, mitochondria and CGI-58, indicating a close association between the key lipolytic effectors in resting skeletal muscle. The colocation of the lipolytic proteins, their independent association with ORO and the PLIN5/ORO colocation were not altered after 60 min of moderate intensity exercise. Further experiments in cultured human myocytes showed that PLIN5 colocation with ORO or mitochondria is unaffected by pharmacological activation of lipolytic pathways. Together, these data suggest that the major lipolytic proteins are highly expressed at the lipid droplet and colocate in resting skeletal muscle, that their localization and interactions appear to remain unchanged during prolonged exercise, and, accordingly, that other post-translational mechanisms are likely regulators of skeletal muscle lipolysis.     

The lipolytic activities of the isolated cell envelope fractions of baker''s yeast

1. The existence of phospholipase and lipase activities in the isolated cell envelopes of baker's yeast was demonstrated. 2. The content of phospholipase was found to be markedly higher than that of lipase. 3. After partial enzymic digestion of the isolated cell envelopes, the bulk of the lipolytic activities was recovered in the sedimentable preparations, which consisted of the fragments of the plasma membrane. 4. During repeated washings, the lipase was completely released from the cell envelopes, as were also the bulk of the lipid components and most of the Mg(2+)-dependent adenosine triphosphatase, an enzyme connected with the plasma membrane. The phospholipase was more firmly bound to the preparation but not so firmly as the external saccharase. 5. These results indicate that the lipolytic enzymes found in the cell envelopes are mostly located in the plasma membrane.     

Triacylglycerol lipase activities in tissues of Antarctic fishes

. The activity of the lipolytic enzyme, triacylglycerol lipase (TAG lipase; E.C. 3.1.1.3) was measured in heart ventricle, liver, oxidative skeletal muscle, and adipose tissue of four species of Antarctic fishes (Chaenocephalus aceratus, Notothenia coriiceps, Trematomus newnesi, Gobionotothen gibberifrons). Estimates of weight-specific activities at 0°C in tissues of the Antarctic fishes were broadly comparable to those reported for temperate-zone fish species assayed at their physiological temperature. Within each species, liver tissue displayed the highest weight-specific activity of TAG lipase in C. aceratus, T. newnesi, and G. gibberifrons, while adipose tissue of N. coriiceps showed the highest activity within that species. Combining these estimates with carefully measured somatic indices of the tissues, permitted assessment of the relative importance of different tissues to overall lipolytic capacity on an organismal level. On this basis, liver tissue displays the greatest capacity of TAG lipase per 100 g body weight in each of the species studied.     

GPIHBP1: an endothelial cell molecule important for the lipolytic processing of chylomicrons

PURPOSE OF REVIEW: To summarize recent data indicating that glycosylphosphatidylinositol-anchored high density lipoprotein-binding protein 1 (GPIHBP1) plays a key role in the lipolytic processing of chylomicrons. RECENT FINDINGS: Lipoprotein lipase hydrolyses triglycerides in chylomicrons at the luminal surface of the capillaries in heart, adipose tissue, and skeletal muscle. The endothelial cell molecule that facilitates the lipolytic processing of chylomicrons has never been clearly defined. Mice lacking GPIHBP1 manifest chylomicronemia, with plasma triglyceride levels as high as 5000 mg/dl. In wild-type mice, GPIHBP1 is expressed on the luminal surface of capillaries in heart, adipose tissue, and skeletal muscle. Cells transfected with GPIHBP1 bind both chylomicrons and lipoprotein lipase avidly. SUMMARY: The chylomicronemia in Gpihbp1-deficient mice, the fact that GPIHBP1 is located within the lumen of capillaries, and the fact that GPIHBP1 binds lipoprotein lipase and chylomicrons suggest that GPIHBP1 is a key platform for the lipolytic processing of triglyceride-rich lipoproteins.     

Inhibition of the reversible deactivation of chicken adipose tissue hormone-sensitive lipase by heat-stable proteins from adipose tissue and skeletal muscle.

The reversible deactivation of chicken adipose tissue hormone-sensitive lipase is catalyzed by a lipase phosphatase. Heat-stable protein preparations from rat epididymal fat pads, chicken adipose tissue, and rabbit skeletal muscle inhibited lipase phosphatase activity. Phosphatase inhibitor preparations from rat adipose tissue did not inhibit the protein kinase-catalyzed activation of hormone-sensitive lipase, whereas inhibitor preparations from rabbit skeletal muscle were contaminated with protein kinase inhibitor.     

The effects of digestive enzymes on characteristics of placental insulin receptor. Comparison of particulate and soluble receptor preparations.

The role of the surrounding membrane structure on the binding characteristics of the insulin receptor was studied by using several digestive enzymes. The effects observed with particulate membrane preparations are compared with those from soluble receptor preparations. beta-Galactosidase and neuraminidase had no effect on insulin binding to either particulate or soluble receptors from human placentae. Exposure to 2 units of phospholipase C/ml increased insulin binding to particulate membranes, but was without effect on the soluble receptor preparation. The increase in binding to particulate membranes was shown to be due to an increase in apparent receptor number. After 5 min exposure to 500 microgram of trypsin/ml there was an increase in insulin binding to the particulate membrane fraction, owing to an increase in receptor affinity. After 15 min exposure to this amount of trypsin, binding decreased, owing to a progressive decrease in receptor availability. In contrast, this concentration of trypsin had no effect on the solubilized receptor preparation. Because of the differential effects of phospholipase C and trypsin on the particulate compared with the solubilized receptor preparations, it is concluded that the effects of these enzymes were due to an effect on the surrounding membrane structure. Changes in receptor configuration due to alterations within the adjoining membrane provide a potential mechanism for mediating short-term alterations in receptor function.     

Adipose triglyceride lipase and the lipolytic catabolism of cellular fat stores

Fatty acids (FAs) are essential components of all lipid classes and pivotal substrates for energy production in all vertebrates. Additionally, they act directly or indirectly as signaling molecules and, when bonded to amino acid side chains of peptides, anchor proteins in biological membranes. In vertebrates, FAs are predominantly stored in the form of triacylglycerol (TG) within lipid droplets of white adipose tissue. Lipid droplet-associated TGs are also found in most nonadipose tissues, including liver, cardiac muscle, and skeletal muscle. The mobilization of FAs from all fat depots depends on the activity of TG hydrolases. Currently, three enzymes are known to hydrolyze TG, the well-studied hormone-sensitive lipase (HSL) and monoglyceride lipase (MGL), discovered more than 40 years ago, as well as the relatively recently identified adipose triglyceride lipase (ATGL). The phenotype of HSL- and ATGL-deficient mice, as well as the disease pattern of patients with defective ATGL activity (due to mutation in ATGL or in the enzyme's activator, CGI-58), suggest that the consecutive action of ATGL, HSL, and MGL is responsible for the complete hydrolysis of a TG molecule. The complex regulation of these enzymes by numerous, partially uncharacterized effectors creates the "lipolysome," a complex metabolic network that contributes to the control of lipid and energy homeostasis. This review focuses on the structure, function, and regulation of lipolytic enzymes with a special emphasis on ATGL.     

Hepatic lipase: a member of a family of structurally related lipases

Partial amino acid sequence of rat hepatic lipase was obtained by gas-phase microsequence analysis of proteolytic fragments. Sequence comparison to bovine lipoprotein lipase and porcine pancreatic lipase reveals a highly conserved region existing among these three physiologically distinct lipolytic enzymes. In a stretch of 36 amino acid residues previously reported for pancreatic lipase (De Caro, J., Boudouard, M., Bonicel, J., Guidoni, A., Desnuelle, P. and Rovery, M. (1981) Biochim. Biophys. Acta 671, 129-138), nineteen residues are identical for all three enzymes, whereas 27 of 36 are identical in rat hepatic lipase and bovine lipoprotein lipase. The fact that this primary structural conservation extends to three different animal species emphasizes the conclusion that these lipolytic enzymes comprise a protein family originating from a common ancestral gene.     

A Two-Step Procedure for Obtaining Highly Purified Particulate Guanylate Cyclase From Rat Lung

Abstract

Cuanylate cyclase (CTP pyrophosphate lyase (cyclizing), EC 4.6.1.21, the enzyme catalyzing the formation of cyclic CMP, from GTP, exists in both soluble and membrane-bound forms.1-6 These enzymes have been implicated as key regulatory components in a variety of biological events such as secretion7-10, and smooth muscle relaxation.11-14 Understanding the role these enzymes play in cellular regulation is predicated upon obtaining purified preparations of guanylate cyclase. Several procedures for purifying the soluble enzyme to apparent homogeneity from a variety of tissues have been reported.15-19 The particulate enzyme has been purified to apparent homogeneity from sea urchin sperm20,21 but, to date, homogeneous preparations of particulate guanylate cyclase from mammalian tissues have not been obtained. Here we report our efforts in purifying particulate guanylate cyclase about 8,200- to 52,000-fold from homogenates of rat lung. Portions of this work have appeared in abstract form.     

Isolation, partial purification and pharmacodynamics of a slow contractile substance in the venom sac extract of the wasp Vespa cincta Fabr

The venom of V. cincta contains acetylcholine (ACh), histamine and 5-hydroxytryptamine (5-HT). Blockers of these agonists did not block completely the hypotensive and smooth muscle contractile activity of venom. On smooth muscle, there was a residual slow contraction. The active substance which produced this slow contraction was separated by solvent extraction, gel filtration and TLC. The purified material (which has been provisionally designated "Vecikinin") lowered cat, rat and guinea pig blood pressure, increased amplitude of cardiac contraction, and increased capillary permeability. Vecikinin contracted several smooth muscle preparations (rat uterus, rat ascending colon, guinea pig ileum, guinea pig colon and rat ileum), while relaxing rat duodenum. Its contractile activity was not lost on boiling, but acid or alkali-boiling reduced its contractile activity. It was inactivated on incubation with chymotrypsin and carboxypeptidase but not with trypsin, pepsin or leucine aminopeptidase. It is a peptide, appears to be of low molecular weight, and could be distinguished from substance P, angiotensin, bradykinin and hornet or wasp kinin.     

Cross-bridge mechanisms underlying the history-dependent properties of muscle spindles and stretch reflexes

The effects of prior movement on the force responses of skeletal muscle are compared with the effects of movement history on the changes in firing rate of muscle spindle receptors. Prior release results in the linearization of the mechanical properties of skeletal muscles, which can be provisionally explained by cross-bridge models of muscular contraction. The history-dependence of responses of muscle spindle receptors in unanesthetized decerebrate preparations appears to result from the kinetics of cycling and noncycling cross-bridges. The results of this comparison indicate that the integration of mechanical properties of muscle and spindle receptor promotes stiffness regulation.     

Human genes involved in lipolysis of plasma lipoproteins: mapping of loci for lipoprotein lipase to 8p22 and hepatic lipase to 15q21

We have used cDNA probes for lipoprotein lipase and hepatic lipase to determine the chromosomal and subchromosomal locations of the human genes for these lipolytic enzymes. Southern blot analysis of genomic DNA from 17 independent mouse-human somatic cell hybrids demonstrated the presence of the gene for human lipoprotein lipase on chromosome 8, whereas the gene for hepatic lipase was on chromosome 15. Regional mapping of the genes by in situ hybridization to human chromosomes indicated that the lipoprotein lipase gene (LPL) resides in the p22 region of chromosome 8, while hepatic lipase gene (HL) resides in the q21 region of chromosome 15. We previously reported, on the basis of nucleotide and amino acid homologies, that these genes are members of a gene family of lipases, and, thus, the present findings indicate that the members of this family are dispersed. The results are also of significance with respect to disorders involving deficiencies of the enzymes. In particular, they suggest that certain rare combined deficiencies of both enzymes do not involve mutations of the structural loci.     

Substrate specificity and kinetic properties of enzymes belonging to the hormone-sensitive lipase family: comparison with non-lipolytic and lipolytic carboxylesterases

We have studied the kinetics of hydrolysis of triacylglycerols, vinyl esters and p-nitrophenyl butyrate by four carboxylesterases of the HSL family, namely recombinant human hormone-sensitive lipase (HSL), EST2 from Alicyclobacillus acidocaldarius, AFEST from Archeoglobus fulgidus, and protein RV1399C from Mycobacterium tuberculosis. The kinetic properties of enzymes of the HSL family have been compared to those of a series of lipolytic and non-lipolytic carboxylesterases including human pancreatic lipase, guinea pig pancreatic lipase related protein 2, lipases from Mucor miehei and Thermomyces lanuginosus, cutinase from Fusarium solani, LipA from Bacillus subtilis, porcine liver esterase and Esterase A from Aspergilus niger. Results indicate that human HSL, together with other lipolytic carboxylesterases, are active on short chain esters and hydrolyze water insoluble trioctanoin, vinyl laurate and olive oil, whereas the action of EST2, AFEST, protein RV1399C and non-lipolytic carboxylesterases is restricted to solutions of short chain substrates. Lipolytic and non-lipolytic carboxylesterases can be differentiated by their respective value of K(0.5) (apparent K(m)) for the hydrolysis of short chain esters. Among lipolytic enzymes, those possessing a lid domain display higher activity on tributyrin, trioctanoin and olive oil suggesting, then, that the lid structure contributes to enzyme binding to triacylglycerols. Progress reaction curves of the hydrolysis of p-nitrophenyl butyrate by lipolytic carboxylesterases with lid domain show a latency phase which is not observed with human HSL, non-lipolytic carboxylesterases, and lipolytic enzymes devoid of a lid structure as cutinase.     

Concanavalin A induced inhibition of 5'-nucleotidase from guinea pig skeletal muscle and bull seminal plasma: a comparative study

Both purified and membrane-bound 5'-nucleotidases (EC 3.1,3.5) from guinea pig skeletal muscle and bull seminal plasma are inhibited by Concanavalin A (Con A). 5'-Nucleotidase purified from skeletal muscle is inhibited by Con A by an apparent uncompetitive process (K'i = 160 nM), while the lectin inhibits the particulate enzyme by an apparent non-competitive process (Ki = K'i = 50 nM). 5'-Nucleotidase purified from bull seminal plasma is inhibited by Con A by an apparent non-competitive process (K'i = Ki = 270 nM), while the membrane-bound enzyme is subjected to a mixed type inhibition by the lectin (K'i greater than Ki; 30 and 14 nM, respectively). The enzyme purified from skeletal muscle exhibits a significant cooperativity in the interaction with Con A. The inhibition of bull seminal plasma particulate 5'-nucleotidase brought about by Con A is not completely reversed by addition of alpha-methyl-D-mannoside.     

The entomopathogen Metarhizium anisopliae can modulate the secretion of lipolytic enzymes in response to different substrates including components of arthropod cuticle

The filamentous fungus Metarhizium anisopliae is a well-characterized, arthropod pathogen used in the biological control of arthropod pests. Studies on the regulation of enzymes related to host infection such as proteases and chitinases have been reported but little is known about regulation of lipolytic enzymes in this fungus. Here we present the effects of different carbon sources such as components of the arthropod cuticle on the secretion of lipolytic enzymes by M. anisopliae. Differences in the induction of lipolytic activity were observed between the several carbon sources tested. Higher activities of lipase or lipase/esterase were found in culture media containing the arthropod integument components chitin and cholesteryl stearate. Several bands of lipolytic activity were also detected in zymograms, thus suggesting an important set of lipolytic enzymes secreted by the fungus. These results show that the fungus can modulate the secretion of lipolytic activity in response to host integument components, thus reinforcing the potential role of these enzymes during M. anisopliae infection.     

Identification and characterization of novel esterases from a deep-sea sediment metagenome

A deep-sea sediment metagenomic library was constructed and screened for lipolytic enzymes by activity-based approach. Nine novel lipolytic enzymes were identified, and the amino acid sequences shared 56% to 84% identity to other lipolytic enzymes in the database. Phylogenetic analysis showed that these enzymes belonged to family IV lipolytic enzymes. One of the lipolytic enzymes, Est6, was successfully cloned and expressed in Escherichia coli Rosetta in a soluble form. The recombinant protein was purified by Ni-nitrilotriacetic affinity chromatography column and characterized using p-nitrophenyl esters with various chain lengths. The est6 gene consisted of 909 bp that encoded 302 amino acid residues. Est6 was most similar to a lipolytic enzyme from uncultured bacterium (ACL67845, 61% identity) isolated from the South China Sea marine sediment metagenome. The characterization of Est6 revealed that it was a cold-active esterase and exhibited the highest activity toward p-nitrophenyl butyrate (C4) at 20°C and pH 7.5.