Influence of apolipoprotein E on soluble and heparin-immobilized hepatic lipase

The effect of human apolipoprotein E (apoE), either alone or in combination with apoC, on the lipolysis of a radiolabeled triglyceride emulsion was studied with hepatic lipase in solution and immobilized on heparin-Sepharose. The soluble hepatic lipase was inhibited, whereas the heparin-immobilized lipase was stimulated by apoE. This stimulation was attenuated by combining apoE with either apoC-II or C-III. The heparin-immobilized lipase demonstrated much less lipolysis of the zwitterionic phosphatidylcholine-stabilized triglyceride emulsion than did the soluble enzyme. This difference was less when the emulsion was stabilized by a nonionic detergent. apoE inhibited lipase activity when assayed under conditions (0.4 M NaCl) of bound enzyme and unbound substrate. Increasing the emulsion apoE content beyond optimum inhibited lipolysis by the immobilized enzyme. Kinetic analysis of phosphatidylcholine-stabilized triglyceride emulsions revealed a significant decrease in immobilized enzyme Km and an increase in Vmax when the emulsion was supplemented with apoE. Distributing the immobilized lipase in clustered aggregates produced more lipolysis than when the same enzyme content was uniformly bound.

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The influence of composition of poly(n-isopropylacrylamide-co-itaconic acid) hydrogel on immobilized Candida rugosa lipase activity

Hemijska industrija ◽

10.2298/hemind0806339m ◽

2008 ◽

Vol 62 (6) ◽

pp. 339-344

Author(s):

Nikola Milasinovic ◽

Melina Kalagasidis-Krusic ◽

Zorica Knezevic-Jugovic ◽

Jovanka Filipovic

Keyword(s):

Mechanical Properties ◽

Itaconic Acid ◽

Lipase Activity ◽

Acid Content ◽

Immobilized Enzyme ◽

In Situ Polymerization ◽

Immobilized Lipase ◽

Candida Rugosa ◽

Candida Rugosa Lipase

The application of lipases as catalysts in chemical reactions has been deterred by the high cost of isolation and purification of enzymes, the instability of their structure when they are isolated from their natural environment, contamination of products with residual protein, their sensitivity to process conditions, etc. These problems could be overcome using immobilized lipases. Immobilization is achieved by fixing enzymes to or within solid supports and as a result a heterogeneous system is obtained. The present paper reports on the immobilization of Candida rugosa lipase in hydrogels based on N-isopropylacrylamide and itaconic acid. Immobilization of lipase is carried out by two different methods. In the first method, lipase is added to the reaction mixture before polymerization and crosslinking (in situ polymerization), while in the second method the synthetized hydrogels are immersed in lipase solution and left to rich the equilibrium swelling. The specific activities of the immobilized lipase were determined in both cases and compared. The amount of the immobilized lipase is higher if the immobilization is carried out by immersing hydrogel in lipase solution. It was observed that in both cases lipase activity increases with an increase of the itaconic acid content up to 10 wt% and thereafter decreases. From the measurements of shear storage moduli (G') it was concluded that the increase of the itaconic acid content decreases the mechanical properties of the hydrogels. SEM analysis confirmed the highly porous structure of hydrogels. It was found that greater pores were achieved when the enzyme was immobilized by in situ polymerization. When the enzyme was immobilized by in situ polymerization the itaconic acid content had not great effect on the mass of the immobilized enzyme, except for the 100/0 sample. On the contrary, for the samples where the enzyme was immobilized by swelling, the increase of the itaconic acid content increases the mass of the immobilized enzyme. Concerning the activity of the immobilized lipase, the swelling degree and mechanical properties of the investigated hydrogels, the best results were performed by the 95/5 hydrogel sample.

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Kinetic Aspects of the Interaction of Blood Clotting Enzymes

Thrombosis and Haemostasis ◽

10.1055/s-0038-1656222 ◽

1965 ◽

Vol 13 (01) ◽

pp. 155-175 ◽

Cited By ~ 4

Author(s):

H. C Hemker ◽

P.W Hemker ◽

E. A Loeliger

Keyword(s):

Kinetic Analysis ◽

Enzyme Kinetics ◽

Blood Clotting ◽

Enzyme System ◽

Enzyme Kinetic ◽

Clotting Time ◽

Standard Methods

SummaryApplication of the methods of enzyme-kinetic analysis to the results of clotting tests is feasible and can yield useful results. However, the standard methods of enzyme kinetics are not applicable without modifications imposed by the peculiarities of the blood-clotting enzyme system. The influence of the following complicating circumstances is calculated :1. Substrate is not present in excess.2. Only relative measures exist for concentrations of substrate or enzymes.3. Enzymes and substrates are often added together.4. Reagents are not pure.5. Clotting-time is our only measure for clotting-velocity.Formulas are deduced, which makes it possible to recognize the effect of these complications.

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