Abstract
Lipoprotein lipase activity has been characterized in solubilized form as a post heparin lipolytic activities. However, membrane-supported enzymes have different reaction velosities than do solubilized enzymes. Recent studies (R. J. Havel, 1973) have shown that human triglyceride rich lipoproteins are the active substrates for the lipoprotein lipase from different sources.
In this study, active catalytic rate on the vascular surface of rat heart for human chylomicron and very low density lipoprotein has been determined.
Materials and Methods
The experimental procedures for determination of membrane supported lipoprotein lipase activity are fundamentally that described by C. J. Fielding, 1974. The heart isolated from male Spragu Dawley rat after overnight fast and was perfused with a modefied recirculating Langendorff apparatus. The components of perfusate are as followed; Krebs-Henseleit bicarbonate buffer gassed with 95% O2 and 5% CO2, 4% v/v of 1.063<d fraction of human plasma, 3.5% w/v of Bovine Serum Albumin (Fraction V) and 3H-labeled human chylomicron or very low density lipoprotein as substrate. The substrate were prepared from both healthy human and the patient with low carboxyl-terminal glutamic acid lipoprotein which was provided by R. J. Havel, UCSF Cardiovascular Research Institute. During the perfusion through the rat heart without arrythmia, 6 or 8 duplicate 0.2ml samples were drawn every 2min. and after extraction of 3H-triglyceride, the kinetic constants, Vmax and Km, were calculated.
Results
The triglyceride of healthy human chylomicron or very low density lipoprotein was obvious during the perfusion through the rat heart. More than 1.0μmol/ml of triglyceride at the begining of the perfusion showed zero order elimination curve, and first order kinetics were shown in less than 0.1μmol/ml of triglyceride concentration. The calculated Vmax and Km were 0.061μmol/min., 0.082m mol in chylomicron and 0.037μmol/min., 0.065m mol in very low density lipoprotein, respectively. On the other hand, any hydrolysis of triglyceride was not observed in the perfusion of low carboxyl-terminal glutamic acid lipoprotein.
Conclusion
Since healthy human triglyceride rich lipoprotein be able to hydrolyze during rat heart perfusion and kinetic constants are similar to previously reported that of rat lipoprotein, it is suggested that the human lipoprotein is an active substrate of the lipoprotein lipase supported on vascular surface of rat heart. It was also demonstrated that abnormal protein component of human plasma, low carboxyl-terminal glutamic acid, inhibits lipoprotein lipase activity in situ.
This experimental model should facilitate to study not only solid-bound enzyme activity but also lipoprotein metabolism on vascular surface of the heart in human subjects.
