Inhibition of lipase activity by low-molecular-weight chitosan

Inhibition of enzymatic activity of lipase (EC 3.1.1.3) from the fungus Candida rugosa and wheat (Triticum aestivum L.) germ by low-molecular-weight chitosan with an average molecular weight of 5.7 kDa in reactions of p-nitrophenyl palmitate cleavage was studied. Preincubation of lipases with chitosan, prior to addition of the substrate to solution, showed that equilibrium during the lipase-inhibitor complex formation was reached within 30 min. The inhibition constants for C. rugosa lipase and wheat germ lipase were 1.4 and 0.9 mM, respectively. The contribution of electrostatic interactions to the complex formation between chitosan and lipases is insignificant.     

Inhibition of lipase activity by low-molecular-weight chitosan

Inhibition of enzymatic activity of lipase (EC 3.1.1.3) from the fungus Candida rugosa and wheat (Triticum aestivum L.) germ by low-molecular-weight chitosan with an average molecular weight of 5.7 kDa in reactions of p-nitrophenyl palmitate cleavage was studied. Preincubation of lipases with chitosan, prior to addition of the substrate to solution, showed that equilibrium during the lipase-inhibitor complex formation was reached within 30 min. The inhibition constants for C. rugosa lipase and wheat germ lipase were 1.4 and 0.9 mM, respectively. The contribution of electrostatic interactions to the complex formation between chitosan and lipases is insignificant.     

Preparation and anticoagulant activity of a low-molecular-weight sulfated chitosan

Synthesis of chitosan sulfates with low molecular weight (M_v 9000–35,000 Da) was carried out by sulfation of low molecular weight chitosan (M_v 10,000–50,000 Da). The oleum was used as sulfating agent and dimethylfornamide as medium. The chitosans were prepared by enzymatic and acidic hydrolysis of initial high molecular weight chitosan as well as by extrusion solid-state deacetylation of chitin. As was shown by FT-IR and NMR-methods and elemental analysis, the sulfation occurred at C-6 and C-3 positions and substitution degree is 1.10–1.63. The molecular weight sulfated chitosan was determined by viscometric method and the Mark–Houwink equation [η]=10⁻⁵ 4.97 M^0.77. Study of anticoagulant activity showed that chitosan sulfates with lowered molecular weight demonstrated a regular increase of anti-Xa activity like heparins.     

Inhibition of Propionibacterium acnes lipase activity by the antifungal agent ketoconazole

The common skin disease acne vulgaris is caused by Propionibacterium acnes. A lipase secreted by this microorganism metabolizes sebum and the resulting metabolites evoke inflammation in human skin. The antifungal drug ketoconazole inhibits P. acnes lipase activity. We previously showed that the drug also inhibits the growth of P. acnes. Thus, ketoconazole may serve as an alternative treatment for acne vulgaris, which is important because the number of antibiotic‐resistant P. acnes strains has been increasing.     

Inhibition of lipoprotein lipase activity by synthetic peptides of apolipoprotein C-III.

In this study we have examined effects of synthetic polypeptide fragments of apoC-III on the kinetic properties of lipoprotein lipase (LPL) activity. Based on the loss of 79% of LPL-inhibitory activity after CNBr cleavage at the N-terminal portion of apoC-III and a systematic search for synthetic peptides with LPL-inhibitory activity spanning the apoC-III sequence, we concluded that the N-terminal domain is the most important in the modulation of LPL activity. In addition, there are multiple attachment sites in apoC-III for its interaction with LPL and these sites reside in the hydrophilic sequences of apoC-III. Probably for this reason the intact apo-CIII exhibited higher inhibitory potential than its peptide components. Based on the deduced inhibition constants derived for the synthetic apoC-III1-79 we concluded that apoC-III is likely to exhibit a physiological role in regulating LPL activity since the derived dissociation constants for the LPL-apoC-III interaction are within the physiological concentration range of plasma apoC-III. In addition, as the synthetic apoC-III1-79 lacks the carbohydrate moiety, we also concluded that the presence of the oligosaccharide in native apoC-III is not essential for its inhibitory activity on LPL. The fact that the I50 (concentration for inhibition of LPL at 50% activity) decreases for apoC-III-1 when assayed in the presence of apoC-II indicated that the activator actually caused an increased affinity between LPL and apoC-III and demonstrated that apoC-III does not compete for the activator site of apoC-II.     

Inhibition of pancreatic lipase B activity by taurodeoxycholate and its reversal by colipase.

In our two-phase reaction system taurodexycholate prevents the adsorption of pancreatic lipase B to the nonaqueous phase. Our data are consistent with a mechanism for this reaction which involves the cooperative formation of an enzyme-(bile salt)4 complex in solution with a dissociation constant of 1.4 X 10(-15)M4. Whereas the free enzyme is readily adsorbed to a bile salt-substrate-covered surface, the complex is not. Thus, the "inhibition" of substrate hydrolysis occurs because enzyme and substrate are separated physically. The protein cofactor, colipase, reverses the inhibitory effects of bile salt by providing a high affinity binding site at the interface for the lipase-(bile salt)4 complex. Steady state and presteady state kinetic data are consistent with the formation of a complex with a 1/1, lipase/colipase, ratio, and a dissociation constant of 0.4 to 2.8 X 10(-9)M. The rate of adsorption of lipase to adsorbed colipase appears to be controlled by diffusion through the unstirred layer with a second order rate constant of 1.3 X 10(6)M-1S-1.     

Esterification activity and stability of Talaromyces thermophilus lipase immobilized onto chitosan

The Talaromyces thermophilus lipase (TTL) was immobilized by different methods namely adsorption, ionic binding and covalent coupling, using various carriers. Chitosan, pre-treated with glutaraldehyde, was selected as the most suitable support material preserving the catalytic activity almost intact and offering maximum immobilization capacity (76% and 91%, respectively). The chitosan-immobilized lipase could be reputably used for ten cycles with more than 80% of its initial hydrolytic activity. Shift in the optimal temperature from 50 to 60 °C and in the pH from 9.5 to 10, were observed for the immobilized lipase when compared to the free enzyme.The catalytic esterification of oleic acid with 1-butanol has been carried out using hexane as organic solvent. A high performance synthesis of 1-butyl oleate was obtained (95% of conversion yield) at 60 °C with a molar ratio of 1:1 oleic acid to butanol and using 100 U (0.2 g) of immobilized lipase. The esterification product is analysed by GC/MS to confirm the conversion percentage calculated by titration.     

Inhibition of lipoprotein lipase activity by sphingomyelin: role of membrane surface structure

We have recently shown that sphingomyelin (SM) strongly inhibits lipoprotein lipase (LPL)-mediated lipolysis in monolayers and emulsion particles. To further evaluate how SM modulates LPL activity on the emulsion surface, the relationship between membrane surface structure and LPL activity was investigated. We measured fluorescence anisotropy of 1-palmitoyl-2-[3-(diphenylhexatrienyl)propionyl]-sn-3-phosphati dylcho line, probing surface acyl chain fluidity, and fluorescence lifetime of N-(5-dimethylaminonaphthalene-1-sulfonyl)dipalmitoylphosphatidylethan olamine in H(2)O and D(2)O buffer, assessing the degree of hydration in the head group region. The results revealed that incorporation of egg SM into triolein-egg phosphatidylcholine emulsions markedly increased acyl chain order and decreased head group hydration of the surface monolayers. In contrast, cholesterol was shown to increase head group hydration despite a strong increase in acyl chain order. The close correlation between the apparent K(m) values of LPL and the degree of head group hydration indicated that LPL interacts with the head group region rather than with the hydrophobic interior of the surface monolayers. However, apparent V(max) did not show a simple correlation with any surface structure, and the finding in which SM had no effect on apparent V(max) of medium-chain triglyceride emulsions suggested that the hydrophobic interaction between acyl chains of SM and triglyceride at the emulsion surface is important for determining the apparent V(max). These results showed conclusively that SM inhibits LPL activity mainly by changing the emulsion surface structure and not by a specific interaction between SM and LPL.     

Preparation of low-molecular-weight chitosan using phosphoric acid

Two types of low degree of polymerisation (DP) chitosan were prepared by homogeneous hydrolysis of chitosan in 85% phosphoric acid at room temperature for 1–6 weeks. The hydrolysates were collected by addition of excess ethanol, and were fractionated by solubility in water. The changes in yields of water-insoluble (higher DP) and water-soluble (lower DP) fractions were determined as a function of hydrolysis time. The hydrolysis proceeded with further deacetylation of chitosan, resulting in degree of deacetylation of more than 90%. The water-insoluble fraction prepared after the hydrolysis for 4 weeks (43% yield) had a weight-average DP ( ) of 16·8, and showed the ‘tendon’ type X-ray diffraction pattern. The water-soluble fraction (12·5% yield) had a of 7·3, and showed the ‘annealed’ type pattern.     

Inhibition of lipoprotein lipase by alkanesulfonyl fluorides

A number of alkanesulfonyl halides (chlorides and fluorides) and esters were synthesized and their effect on the activity of lipoprotein lipase (LPL) was studied. Sulfonyl fluorides proved to be efficient inhibitors of LPL when the enzyme was incubated with a 10-fold molar excess of the inhibitors in a buffer containing bile salts (deoxycholate). Hexadecane- and dodecanesulfonyl fluorides caused 50% inhibition of LPL activity at concentrations of 10 to 20 μM.     

Inhibition of lipase activities by basic polysaccharide

Basic polysaccharide strongly inhibited the hydrolysis of trioleoylglycerol (TO) emulsified with phosphatidylcholine and taurocholate by either pancreatic lipase or carboxylester lipase. DEAE-Sephadex dose-dependently inhibited the hydrolysis of TO by pancreatic lipase and carboxylester lipase; however, carboxymethyl-Sephadex and Sephadex G-50 did not inhibit the hydrolysis. Polydextrose (PD), a soluble polysaccharide, was a very weak inhibitor of pancreatic lipase. However, when a basic group, a DEAE group, was attached to PD, lipase inhibition by DEAE-PD was increased, and this was dependent on the substitution ratio of DEAE groups. The number of positive charges per PD molecule is important in lipase inhibition. Similar substitution effects were observed with other basic groups, such as piperidinoethyl and 3-triethylamino-2-hydroxypropyl. The natural basic polysaccharide, chitosan, also inhibited pancreatic lipase activity. Gel-filtration experiments suggested that DEAE-PD did not bind strongly to pancreatic lipase. The effect of DEAE-PD on TO hydrolysis by pancreatic lipase was studied using various emulsifiers: DEAE-PD (50 microg/ml) did not inhibit the hydrolysis of TO emulsified with arabic gum, phosphatidylserine, or phosphatidic acid. In vivo, oral administration of DEAE-PD to rats reduced the peak plasma triacylglycerol concentration and increased fecal lipid excretion. These results suggest that basic polysaccharide is able to suppress dietary fat absorption from the small intestine by inhibiting pancreatic lipase activity.     

Inhibition of hepatic lipase activity impairs chylomicron remnant-removal in rats

[4-14C]Cholesteryl oleyl ether-labeled chylomicron remnants were injected into rats which received a specific goat antibody against rat hepatic lipase or a control serum. Chylomicron remnant cholesterol ether disappeared from circulation with a significantly higher half-life (2-fold) in antibody-treated rats than in controls (P less than 0.001). Recovered radioactivity in the liver was 2-fold lower in antibody-treated rats (22.8% (n = 6) vs. 45% (n = 4) P less than 0.01). These results clearly show that hepatic lipase may strongly promote chylomicron remnant cholesterol ether uptake by the liver.     

Inhibition of amyloidosis using low-molecular-weight heparins

BACKGROUND: Amyloid diseases are characterized by the tissue deposition of extracellular proteinaceous material, which results in organ dysfunction and death. Colocalization of heparan sulfate (HS) proteoglycans to amyloid deposits suggests that they may be an early event in amyloid formation and play an important role in fibril formation. Structural analysis has demonstrated that HS interacts with amyloidogenic proteins resulting in structural changes that allow for an increase in beta-sheet content, possibly enhancing fibrillogenesis. Recent studies have shown that small-molecule anionic sulfonates or sulfates can arrest inflammation-associated (AA) amyloid induction. MATERIALS AND METHODS: In the present study, we examine the effect of low-molecular-weight heparins (LMWHs) on the development of amyloid in the mouse model of AA amyloid. In addition, in vitro fibril formation assays were performed to determine the effect of LMWHs on fibrillogenesis. RESULTS: Injection of mice with clinically relevant doses of LMWHs (enoxaparin and dalteparin) demonstrated a reduction in AA amyloid deposition. These compounds were capable of arresting the progression of AA amyloid and eventually resulting in regression of the amyloid deposits. In vitro analysis indicated that LMWHs prevented AA and Abeta peptide fibril formation by impeding the structural changes necessary for fibril formation. CONCLUSIONS: Our findings suggest that the LMWHs may provide beneficial effects against the development of amyloidoses, including Alzheimer's disease.     

Obtaining and study of monosaccharide derivatives of low-molecular-weight chitosan

The possibility of obtaining monosaccharide derivatives of low-molecular-weight chitosan with the use of the Maillard reaction was studied. Chitosan derivatives (molecular weight, 24 and 5 kDa) obtained with glucosamine, N-acetyl galactosamine, galactose, and mannose with a substitution degree of 4-14% and a yield of 60-80% were obtained. Some physicochemical and biological properties of these derivatives were studied. We showed that monosaccharide derivatives of low-molecular-weight chitosan exhibited antibacterial activity. Chitosan at a concentration of 0.01% caused 100% death of bacteria B. subtilis and E. coil. The strongest antibacterial effect was exhibited by 24-kDa derivatives: only 0.02-0.08% of cells survived. These derivatives were two orders of magnitude more effective than the 5-kDa chitosan modified with galactose.     

Obtaining and study of monosaccharide derivatives of low-molecular-weight chitosan

The possibility of obtaining monosaccharide derivatives of low-molecular-weight chitosan with the use of the Maillard reaction was studied. Chitosan derivatives (molecular weight, 24 and 5 kDa) obtained with glucosamine, N-acetyl galactosamine, galactose, and mannose with a substitution degree of 4–14% and a yield of 60–80% were obtained. Some physicochemical and biological properties of these derivatives were studied. We showed that monosaccharide derivatives of low-molecular-weight chitosan exhibited antibacterial activity. Chitosan at a concentration of 0.01% caused 100% death of bacteria B. subtilis and E. coli. The strongest antibacterial effect was exhibited by 24-kDa derivatives: only 0.02–0.08% of cells survived. These derivatives were two orders of magnitude more effective than the 5-kDa chitosan modified with galactose.     

Galactosylated derivatives of low-molecular-weight chitosan: production and properties

Chitosan, a binary heteropolysaccharide consisting of 2-acetamide-2-deoxy-beta-D-glucopyranose and 2-amino-2-deoxy-beta-D-glucopyranose residues linked in different proportions via beta-glycosidic bonds. The presence of a primary amino group in the chitosan structure allows for the synthesis of various derivatives. The procedure of obtaining activated N-hydroxysuccinimide esters with the use of lactobionic acid was applied to obtain galactosylated derivatives of low-molecular-weight chitosan with a substitution degree varying from 8 to 23%. The properties of these derivatives (viscosity, solubility, and biodegradability) were studied. These derivatives are well soluble at pH values greater than the acidity constant of amino groups of chitosan (6.5). Broadening the pH range towards increase and the presence of galactose residues allows these derivatives to be used in working with biological objects.