Stimulated Lipase Activity Research Articles

Modulating the performance of lipase-hydrogel microspheres in a “micro water environment”

In our previous work, we successfully stimulated lipase activity in an anhydrous reaction system using porous polyacrylamide hydrogel microsphere (PPAHM) as a carrier of lipase and free water. However, the effect of the existence state and content of water in lipase-porous polyacrylamide hydrogel microsphere (L-PPAHM) on the interfacial activation remained unclear. In this work, L-PPAHM with different water contents were obtained by water mist rehydration and were used to catalyze the synthesis of conjugated linoleic acid ethyl ester (CLA-EE). The results revealed that there were three existence states of water in L-PPAHM: bound water, semi-bound water and free water, and free water provided the “micro water environment” for the interfacial activation of lipase. The reusability of L-PPAHM with different water contents showed that the activity and stability of L-PPAHM could be achieved by varying the water content of L-PPAHM. The proportion of free water in L-PPAHM increased, and the activity of L-PPAHM increased, but the strength of hydrogen bond interaction between PPAHM and lipase weakened, resulting in the decrease of stability. L-PPAHM with 2/3 of water absorption could ensure sufficient immobilized lipase activity and stability, and its water absorption property could reduce the free water generated during esterification, thus increasing the yield of CLA-EE.

Purification and Characterization of Lipase Produced by Leuconostoc mesenteroides Subsp. mesenteroides ATCC 8293 Using an Aqueous Two-Phase System (ATPS) Composed of Triton X-100 and Maltitol.

Purification of lipase produced by L. mesenteroides subsp. mesenteroides ATCC 8293 was conducted for the first time using a novel aqueous two-phase system (ATPS) composed of Triton X-100 and maltitol. The partitioning of lipase was optimized according to several parameters including pH, temperature, and crude load. Results showed that lipase preferentially migrated to the Triton X-100 rich phase and optimum lipase partitioning was achieved in ATPS at TLL of 46.4% and crude load of 20% at 30 °C and pH 8, resulting in high lipase purification factor of 17.28 and yield of 94.7%. The purified lipase showed a prominent band on SDS-PAGE with an estimated molecular weight of 50 kDa. The lipase was stable at the temperature range of 30–60 °C and pH range of 6–11, however, it revealed its optimum activity at the temperature of 37 °C and pH 8. Moreover, lipase exhibited enhanced activity in the presence of non-ionic surfactants with increased activity up to 40%. Furthermore, results exhibited that metals ions such as Na+, Mg2+, K+ and Ca2+ stimulated lipase activity. This study demonstrated that this novel system could be potentially used as an alternative to traditional ATPS for the purification and recovery of enzymes since the purified lipase still possesses good process characteristics after undergoing the purification process.

Abstract 537: Characterization of the Lipase Stimulating Domain for Apolipoprotein A-V and the Development of a Therapeutic Peptide for the Treatment of Hypertriglyceridemia

The impaired clearance of plasma triglycerides is a major cause of clinical hypertriglyceridemia, a condition which affects about one third of adults in the G7 countries and is a risk factor for a number of diseases, including cardiovascular disease, cancer and pancreatitis. The hydrolysis and removal of triglycerides from the plasma is dependent on cell surface lipoprotein lipase (LpL) and its enzymatic activity is in turn regulated by specific apolipoproteins; apoC-III, which acts as a potent inhibitor and apoC-II and apoA-V, which act as stimulators. Their importance in hypertriglyceridemia is underscored by GWAS analyses, which have identified the APOA1/C3/A4/A5 gene cluster (chromosome 11q23) as an important determinant of dylipidemia. ApoA-V is of particular interest since its physiological concentration (3.8 nM) is 10,000x lower than that of apoC-II and the other major apolipoproteins. To investigate the influence of different lipoproteins on lipase kinetics we have developed a fluorescence-based assay that uses a fluorogenic triglyceride analog (EnzChek®) in a microplate format. Here we report that a short alpha-helical amphipathic peptide (AV199-232) corresponding to apoA-V residues 199-232 exhibit; i) lipid-binding, ii) heparin-binding and iii) lipase-stimulating activities. Structural characterization and residue substitution studies have been employed to map these three activities to three separate discrete domains within AV199-232. Enzyme kinetic analysis with pure LpL indicates that AV199-232 can overcome the inhibition of LpL by apoC-III and stimulate enzyme activity by reducing its Km 8-fold. Similar increases in lipase activity are also observed with live THP-1 monocytes and HepG2 cells that express hepatic lipase. The influence of AV199-232 in cell culture is saturable, dependent on the presence of cell surface lipase, and enzymatic activation can be sustained for at least 2 hrs. We postulate that apoA-V and AV199-232 stimulate lipase activity through an allosteric mechanism by binding to lipases with high affinity near their catalytic site to affect enzyme kinetics.

Anti- and Pro-Lipase Activity of Selected Medicinal, Herbal and Aquatic Plants, and Structure Elucidation of an Anti-Lipase Compound

Plants that help in slowing down the digestion of triacylglycerols (TAGs) in the pancreas and small intestine of humans play an important role in the reduction of obesity. On the other hand, there may be plants or plant parts that stimulate intestinal lipolytic activity, thus contributing to greater TAG assimilation. The aim of this study was to evaluate the aqueous methanolic extracts of ninety eight (98) medicinal, herbal and aquatic plant materials from Malaysia for their effect on porcine pancreatic lipase (PPL) activity and to identify the structure of an anti-lipase compound from one of the sources. The degree of inhibition was also quantified as relative to orlistat activity against PPL (orlistat equivalents). Results revealed that while 19.4% of the extracts were found to have anti-lipase activity ≥80%, 12% were actually found to promote PPL activity. Twenty two percent (22.4%) exhibited moderate inhibition (41%–80%) and 2% were neutral toward PPL activity. The ripe fruit of Averrhoa carambola and the leaves of Archidendron jiringa (Jack) I.C Nielsen L. (jering), Cynometra cauliflora (nam-nam) and Aleurites moluccana (L.) Willd (candle nut/buah keras) had the highest (100%) anti-lipase activity and are equivalent to 0.11 µg orlistat/mL. Plants that stimulated lipase activity included Pimpinella anisum L. (aniseed/jintan manis), activating the enzyme by 186.5%. Kaempferol 3-O-rhamnoside was isolated from the ethyl acetate fraction of C. cauliflora leaves and found to be an active lipase inhibitor. The structure was elucidated using 1H-NMR, 13C-NMR and 2D-NMR analyses.

Application of a combined approach involving classical random mutagenesis and metabolic engineering to enhance FK506 production in Streptomyces sp. RM7011

FK506 production by a mutant strain (Streptomyces sp. RM7011) induced by N-methyl-N'-nitro-N-nitrosoguanidine and ultraviolet mutagenesis was improved by 11.63-fold (94.24mg/l) compared to that of the wild-type strain. Among three different metabolic pathways involved in the biosynthesis of methylmalonyl-CoA, only expression of propionyl-CoA carboxylase (PCC) pathway led to a 1.75-fold and 2.5-fold increase in FK506 production and the methylmalonyl-CoA pool, respectively, compared to those of the RM7011 strain. Lipase activity of the high FK506 producer mutant increased in direct proportion to the increase in FK506 yield, from low detection level up to 43.1U/ml (12.6-fold). The level of specific FK506 production and lipase activity was improved by enhancing the supply of lipase inducers. This improvement was approximately 1.88-fold (71.5mg/g) with the supplementation of 5mM Tween 80, which is the probable effective stimulator in lipase production, to the R2YE medium. When 5mM vinyl propionate was added as a precursor for PCC pathway to R2YE medium, the specific production of FK506 increased approximately 1.9-fold (71.61mg/g) compared to that under the non-supplemented condition. Moreover, in the presence of 5mM Tween 80, the specific FK506 production was approximately 2.2-fold (157.44mg/g) higher than that when only vinyl propionate was added to the R2YE medium. In particular, PCC expression in Streptomyces sp. RM7011 (RM7011/pSJ1003) together with vinyl propionate feeding resulted in an increase in the FK506 titer to as much as 1.6-fold (251.9mg/g) compared with that in RM7011/pSE34 in R2YE medium with 5mM Tween 80 supplementation, indicating that the vinyl propionate is more catabolized to propionate by stimulated lipase activity on Tween 80, that propionyl-CoA yielded from propionate generates methylmalonyl-CoA, and that the PCC pathway plays a key role in increasing the methylmalonyl-CoA pool for FK506 biosynthesis in RM7011 strain. Overall, these results show that a combined approach involving classical random mutation and metabolic engineering can be applied to supply the limiting factor for FK506 biosynthesis, and vinyl propionate could be successfully used as a precursor of important methylmalonyl-CoA building blocks.

Lipase activity in Thermus thermophilus HB8: Purification and characterization of the extracellular enzyme

In this study, the lipolytic activity of Thermus thermophilus HB8 was examined. The addition of various oils increased the production of extracellular lipolytic activity, while a combination of olive oil and glucose increased both extracellular and intracellular lipolytic activity. The oxygen transfer rate had a significant influence on both biomass and production of extra- or intra-cellular lipolytic activity. The formation of white halos due to the hydrolysis of oleic acid ester (Tween 80) in agar plates containing Nile Blue and the formation of Ca2+-oleate indicated the secretion of lipase. When the cell-free supernatant of cells grown in basal reach medium or the corresponding intracellular extract were electrophoresed under denatured and renatured conditions, using α-naphthyl acetate and Fast Blue RR, major bands at 56 kDa or 62 and 32 kDa were observed, respectively. The 56 kDa extracellular enzyme was partial purified and characterized. Its peak of activity occurred at 80°C and pH 7.0, while the T1/2 was 1 h at 100°C. The Km of the partial purified enzyme was 1 mM and the Vmax was 0.044 U/mL/min when using p-nitrophenyl laurate as substrate. The presence of Ca2+ and Hg2+ stimulated lipase activity, whereas Zn2+, Co2+, or EDTA inhibited lipase activity. The highest activity was observed in the presence of coconut oil and p-nitrophenyl laurate (pNPL). Purified lipase was the most stable in the presence of various organic solvents, such as pentanol, chloroform and n-dodecane. Because of the superior thermostability and stability in the presence of organic solvents of T. thermophilus extracellular lipase, this lipase holds great promise for use in industrial applications.

Biochemical properties and potential applications of an organic solvent-tolerant lipase isolated from Bacillus cereus BF-3

An organic solvent-tolerant strain producing lipase was isolated. The optimal pH and temperature were 8.0 and 34°C, respectively. The lipase was stable at 50°C after 1 h incubation at pH 6 to 8. BF-3 lipase was very stable when incubated with methanol, ethanol and toluene below 30% (v/v) but was stability reduced drastically above 50%. While K + , Na + , Mg 2+ and Ba 2+ (1 mMol/L; 3 mMol/L) stimulated lipase activity, EDTA inhibited its activity indicating that the lipase from Bacillus cereus BF-3 was a metalloenzyme. Key words: Organic, solvent, tolerant, lipase, Bacillus cereus BF-3, biochemical, properties.

Chapter 7 - Physical, Chemical, and Biological Properties of Wonder Kelp— Laminaria

Laminaria is a kelp that finds its place in the brown algae family. It has been an area of study for past many years, and its wonderful biological properties have always attracted medical professionals and researchers to explore more and more from this wonder kelp. The constituents of Laminaria include iodine, potassium, magnesium, calcium and iron. Iodine compounds, TEA-hydroiodide in particular, are great lipolytic agents as they stimulate lipase activity. Laminarins on the other hand are used as a tumor angiogenic blocker. This genus of the kelps is also rich in algin, a high molecular weight polysaccharide that forms viscous colloidal solutions or gels in water leading to the use of kelp derivatives as bulk laxatives. It has great applications in cosmeceutical science, as well as some antibacterial properties have also been assigned to Laminaria. A deeper insight into the physical, biological, and chemical properties of this wonder kelp would lead to further exploitation of Laminaria for medicinal and cosmeceutical purpose.

Characterization of an extracellular alkaline lipase from Pseudomonas mendocina M‐37

A strain of Pseudomonas mendocina producing extracellular lipase was isolated from soil. The bacterium accumulates lipase in culture fluid when grown aerobically at 30 °C for 24 h in a medium composed of olive oil (1%) as substrate. Pseudomonas mendocina lipase was optimally active at pH 9.0, temperature of 50 °C and was found to be stable between pH 7.0-11.0. The lipase was inhibited by detergents such as SDS and Tween-80. The enzyme was stable in various organic solvents tested with maximum stability in chloroform followed by toluene and exhibited 1-3 regiospecificity for hydrolytic reaction. This lipase was capable of hydrolyzing a variety of lipidic substrates and is mainly active towards synthetic triglycerides and fatty acid esters that possess a butyryl group. Metal ions like Mg(2+), Ca(2+) and Na(+) stimulated lipase activity, whereas, Cu(2+), Mn(2+) and Hg(2+) ions caused inhibition.

Dependence of Phospholipase D1 Multi-monoubiquitination on Its Enzymatic Activity and Palmitoylation

Phospholipase D (PLD) is an important lipase in many cellular processes, including vesicular trafficking, cell survival, and cell migration. In the present study, we show that PLD1, but not PLD2, is posttranslationally modified by multi-monoubiquitination. Intriguingly, suppression of lipase activity either by mutation of the HKD motif (PLD1 H896R, K898R, or D903A) or the phosphatidylinositol 4,5-bisphosphate binding motif (PLD1 R691G,R695G) or through use of PLD-selective inhibitors impaired the ubiquitination of PLD1, although stimulation of lipase activity by phorbol 12-myristate 13-acetate did not enhance its ubiquitination. A palmitoylation-deficient mutant PLD1 allele, which exhibits altered patterns of vesicular trafficking, had significantly lower levels of monoubiquitination. In addition, the expression of ubiquitin-fused PLD1 induced aberrantly enlarged vesicles partially co-localized with the Golgi complex but not with early endosomes. The altered localization was reduced by the K898R mutation, suggesting a role of multi-monoubiquitination in PLD1 subcellular localization. Surprisingly, the degradation of PLD1, but not of PLD1 K898R or PLD2, was blocked by inhibitors of proteasomes but not by inhibitors of lysosomes or other proteases, suggesting a role of the ubiquitination in proteasomal degradation of PLD1. In summary, our studies show that PLD1, but not PLD2, is multi-monoubiquitinated. The ubiquitination modification might represent a novel regulatory mechanism in PLD1 functioning, particularly in the context of subcellular trafficking between different membrane compartments.

Biocatalytic Properties of Lipase from Walnut Seed (Juglans regia L.)

AbstractLipase (E.C. 3.1.1.3) from walnut seed was purified 28.6‐fold with 31% yield using Sephadex G‐100 gel chromatography. Olive oil served as good substrate for the enzyme. The optimum pH and temperature were 9.0 and 70 °C, respectively. The lipase was stable between 30 and 80 °C for 5 min. Km and Vmax values were determined as 48 mM and 23.06 × 10−3 U/min mg for triolein as substrate. Lipase activity was slightly reduced by Cu2+, Ca2+, Hg2+, Mn2+, and Ni2+ ions, while Mg2+ and Zn2+ had no effects. Anionic surfactant sodium dodecyl sulfate stimulated lipase activity while non‐ionic surfactants Tween‐80 and Triton X‐100 had negligible effects on enzymatic activity. The enzyme activity was not affected by 50 mM urea and thioacetamide. Potassium ferricyanide, n‐bromosuccinamide and potassium cyanide reduced the enzyme activity. The enzyme showed a good stability in organic solvents, the best result being in n‐hexane (113% residual activity). The activity of dialysate was maintained approximately 80% for 1 year at −20 °C.

Isolation of a lipase-secreting yeast for enzyme production in a pilot-plant scale batch fermentation

The production of lipase by twenty-nine yeasts isolated from the phylloplane of Hibiscus rosa-sinensis was evaluated. The highest lipase producers were Pseudozyma hubeiensis HB85A, Debaryomyces occidentalis-like HB83 and Cryptococcus sp. HB80. P. hubeiensis HB85A batch fermentations were carried out in a bioreactor and lipase production improved 3.2-fold as compared to flask submerged cultures. The production process was significantly reduced from 48 h (in flasks) to 18 h (in the bioreactor). The better hydrolytic activity was achieved with C16 p-nitrophenyl ester. Maximal activity was observed at pH 7.0, the optimum temperature was 50 °C at pH 7.0 and the enzyme was stable at 30 and 40 °C. The lipolytic activity was stimulated by Mg 2+, K + and Ba 2+ salts and EDTA and slightly inhibited by Ca 2+ salts. Non-ionic detergents such as Triton X-100, Tween 80 and Tween 20 strongly stimulated lipase activity, whereas SDS inhibited it. The lipase was stable in iso-octane and hexane at 80%.

Regulation of lipolytic activity by long-chain acyl-coenzyme A in islets and adipocytes

Intracellular lipolysis is a major pathway of lipid metabolism that has roles, not only in the provision of free fatty acids as energy substrate, but also in intracellular signal transduction. The latter is likely to be particularly important in the regulation of insulin secretion from islet beta-cells. The mechanisms by which lipolysis is regulated in different tissues is, therefore, of considerable interest. Here, the effects of long-chain acyl-CoA esters (LC-CoA) on lipase activity in islets and adipocytes were compared. Palmitoyl-CoA (Pal-CoA, 1-10 microM) stimulated lipase activity in islets from both normal and hormone-sensitive lipase (HSL)-null mice and in phosphatase-treated islets, indicating that the stimulatory effect was neither on HSL nor phosphorylation dependent. In contrast, we reproduced the previously published observations showing inhibition of HSL activity by LC-CoA in adipocytes. The inhibitory effect of LC-CoA on adipocyte HSL was dependent on phosphorylation and enhanced by acyl-CoA-binding protein (ACBP). In contrast, the stimulatory effect on islet lipase activity was blocked by ACBP, presumably due to binding and sequestration of LC-CoA. These data suggest the following intertissue relationship between islets and adipocytes with respect to fatty acid metabolism, LC-CoA signaling, and lipolysis. Elevated LC-CoA in islets stimulates lipolysis to generate a signal to increase insulin secretion, whereas elevated LC-CoA in adipocytes inhibits lipolysis. Together, these opposite actions of LC-CoA lower circulating fat by inhibiting its release from adipocytes and promoting fat storage via insulin action.

Properties of a thermostable extracellular lipase from Bacillus megaterium AKG‐1

An extracellular lipase isolated from Bacillus megaterium AKG-1 had an optimum activity at 55 degrees C/pH 7.0. It retained 100% activity at 50 degrees C for 30 min with a half life of 30 min at 70 degrees C. A 20-70% increase in lipase activity was observed in presence of acetone (20% v/v), DMSO (20% v/v) and isopropanol (10% v/v). The enzyme activity was 92, 98 and 107% after 24 h, on treatment with 10% (v/v) acetone, benzene and isopropanol respectively. Deoxycholic acid, sodium deoxycholate, lithocholic acid, rhamnolipid, Brij 52 and cholic acid stimulated the lipase activity by 76, 36, 24, 24, 23.6 and 13%, respectively. Addition of reducing agents like sodium sulphite, sodium metabisulphite and L-cysteine-HCl, at 10 mM concentration stimulated lipase activity by 127, 146 and 150% respectively. The lipase appeared to show enantioselectivity in hydrolyzing racemic 3-acetoxy-beta-lactam as it hydrolyzed only the (+) enantiomer.

High-yield purification of an organic solvent-tolerant lipase from Pseudomonas sp. strain S5

An organic solvent-tolerant S5 lipase was purified by affinity chromatography and anion exchange chromatography. The molecular mass of the lipase was estimated to be 60 kDa with 387 purification fold. The optimal temperature and pH were 45 °C and 9.0, respectively. The purified lipase was stable at 45 °C and pH 6–9. It exhibited the highest stability in the presence of various organic solvents such as n-dodecane, 1-pentanol, and toluene. Ca 2+ and Mg 2+ stimulated lipase activity, whereas EDTA had no effect on its activity. The S5 lipase exhibited the highest activity in the presence of palm oil as a natural oil and triolein as a synthetic triglyceride. It showed random positional specificity on the thin-layer chromatography.

Purification and characterization of a regiospecific lipase from Aspergillus terreus.

Aspergillus terreus lipase was purified to homogeneity with 18.0% yield. The specific activity of the enzyme increased from 20.80 to 250 U/mg of protein. Ion exchange on Q-Sepharose was highly effective in the purification process. The molecular mass of the purified enzyme was 41+/-1 kDa as determined by SDS/PAGE. The purified lipase showed excellent temperature tolerance (15-90 degreesC) and was highly thermostable, retaining 100% activity at 60 degreesC for 24 h. It showed good pH tolerance (3.0-12.0) and was stable over a pH range of 4.0-10.0 for 24 h. The activity of the enzyme was inhibited by ionic detergents, whereas non-ionic detergents stimulated enzyme activity. Mg2+ and Ca2+ ions stimulated lipase activity, whereas Co2+, Cu2+, Ni2+ and Fe3+ ions caused inhibition. The enzyme was unaffected by the metal chelator EDTA or by 2-mercaptoethanol and potassium ferrocyanide. At a concentration of 100 microM, 3,4-dichloroisocoumarin caused weak inhibition with 40% loss of activity, but diethyl p-nitrophenyl phosphate at the same concentration strongly inhibited enzyme activity (98.12% loss of activity), confirming that the A. terreus lipase is a serine hydrolase. The lipase was highly active on pig fat (151% relative activity) and groundnut oil (103% relative activity) and least active on kusum oil (18% relative activity). Extensive dialysis did not affect enzyme activity up to 168 h, suggesting the absence of any dialysable cofactor in the enzyme. The A. terreus lipase retained significant activity on freeze-drying and had a shelf-life of more than 6 months at room temperature. The A. terreus lipase exhibited 1,3-regiospecificity and was stable in various organic solvents.

Stimulation of pancreatic lipase activity by saponins isolated from Medicago sativa L.

Saponins isolated from Medicago sativa L. appeared to stimulate lipolytic activity and did not influence amylolytic or proteolytic activity of Neopancreatinum (extract of pancreatic enzymes: lipase, proteases and amylases). Saponins isolated from the aerial part of Medicago sativa L. were demonstrated to stimulate lipase activity more effectively than saponins separated from the root of this plant. When saponins were treated with 0.02 M HCl at 37 degrees C for 1 hour their lipase stimulation ability did not change. The saponins stimulated Neopancreatinum lipolytic activity more intensively in the presence of sodium cholate.

Partial purification of a diacylglycerol lipase from bovine aorta.

A diacylglycerol (DG) lipase has been purified from a soluble subcellular fraction of bovine aorta by (NH4)2SO4 precipitation in the presence of 5.0% (w/v) Triton X-100, followed by chromatography on DEAE-Sephacel, heparin-Sepharose and octyl-Sepharose in the presence of either CHAPS or Triton X-100 detergents. Under basal conditions, the hydrolysis of a short-chain [3H]dioctanoylglycerol ([3H]diC8) substrate was much greater than that of a long-chain 1-[1-14C]palmitoyl-2-oleoyl-sn-glycerol (1-[14C]POG) substrate. Lipase activity measured with 1-[14C]POG was markedly enhanced by Triton X-100. In the presence of 0.1% Triton X-100, specific enzyme activities in the octyl-Sepharose fraction determined with 1-[14C]POG or 1-stearoyl-2-[1-14C]-arachidonoyl-sn-glycerol as substrates were the same as that measured with [3H]diC8. MgCl2 (5mM) or CaCl2 (2 mM) also selectively stimulated lipase activity (up to 10-13-fold) measured with the long-chain (1-[14C]POG) substrate only. The increase in relative specific activity in the octyl-Sepharose fraction was 60-fold and 155-fold, based on hydrolysis of [3H]diC8 and 1-[14C]POG (+ Triton X-100), respectively. Unlabelled diC8 was a competitive inhibitor of 1-[14C]POG hydrolysis, suggesting that a single lipase hydrolyses both the short-chain and long-chain DG substrates; selective stimulatory effects of non-ionic detergents and bivalent cations on the hydrolysis of 1-[14C]POG may be due to effects on the physical properties of the substrate preparation. Monoacylglycerol lipase, DG kinase and cholesterol esterase activities could not be detected in the partially purified lipase preparation.

In vivo effect of free fatty acids on the specific binding of glucocorticosteroids to corticosteroid binding globulin and liver receptors in immature rats

Stimulating lipase activity with heparin (200 IU/kg b.w.) increased the plasma free fatty acid (FFA) concentration of immature rats (15 days). The effect of this elevated FFA concentration on glucocorticoid binding to corticosteroid binding globulin (CBG), and liver cytosol glucocorticoid receptor (GR), was analyzed. The plasma FFA concentration increased 2-fold, 10 minutes ( P < 0.001), 20 minutes ( P < 0.01), and 60 minutes ( P < 0.01) post-heparin. The corticosterone (B) and progesterone concentrations were unchanged 60 minutes post-injection. The binding activity of immature rat CBG for B dropped 50% ( P < 0.001) 60 minutes post-heparin injection, decreased B binding and increased plasma FFA were correlated ( r = −0.8 ). The decreased B binding resulted from a 2-fold decrease in the apparent number of CBG binding sites; the affinity constant (K a) remained unchanged. The liver cytosol endogenous FFA content of immature rats was also increased 2-fold, 60 minutes after heparin-induced lipolysis. The increased cytosol FFA, with no significant change in glucocorticoid, was accompanied by a significant decrease in dexamethasone binding to liver cytosol glucocorticoid receptor. The decrease resulted from a significantly lower apparent K a for dexamethasone and fewer receptor binding sites (n). There was a good inverse correlation between K a ( r = −0.93 ) and n ( r = −0.90 ) and the increased liver cytosol FFA content. Thus the higher plasma FFA induced in vivo by lipase activation or a standard FFA mixture probably causes conformational changes in CBG and GR, reducing glucocorticoid binding to immature rat CBG and liver GR.

Protein kinase C and progesterone-induced maturation in Xenopus oocytes

Though progesterone-induced maturation has been studied extensively in Xenopus oocytes, the mechanism whereby the prophase block arrest is released is not well understood. The current hypothesis suggests that a reduction in cAMP and subsequent inactivation of cAMP-dependent protein kinase is responsible for reentry into the cell cycle. However, several lines of evidence indicate that maturation can be induced without a concomitant reduction in cAMP. We show that the mass of diacylglycerol in whole oocytes and plasma membranes decreases 29% and 10% respectively, within the first 15 sec after the addition of progesterone. Diacylglycerol in plasma membranes further decreased 59% by 5 min. We also show that the protein kinase C inhibitors sphingosine and staurosporine can induce oocyte maturation. In addition, the synthetic diglyceride, DiC8, and microinjected PKC can inhibit or delay progesterone-induced maturation. These results together suggest that a transient decrease in protein kinase C activity may regulate entry into the cell cycle. The mechanism whereby DAG is decreased in response to progesterone is unclear. Initial studies show that progesterone leads to a decrease in IP3 suggesting that progesterone may act by reducing the hydrolysis of PIP2. On the other hand, progesterone caused a decrease in the amount of [3H]arachidonate labelling in DAG during the same time suggesting that progesterone may stimulate lipase activity. The relationship between postulated changes in the PKC pathway and those hypothesized for the PKA pathway are discussed.