The isolation and characterization of lipoprotein lipase of the rainbow trout
1. Rainbow trout (Salmo granderi Richardson) was shown to possess lipoprotein lipase which was released into the circulation after intravenous injection of heparin, reaching maximum activity after 40 minutes. 2. Trout post-heparin plasma LPL showed maximum activity at a temperature of 35°C and pH 8.2. Sodium chloride, protamine sulphate, sodium deoxycholate and heparin showed inhib similar to that of human LPL (EC 126.96.36.199) and distinguishes it from hepatic salt-resistant lipase (SRL) which was not detected in trout post-heparin plasma. 3. Heparin-Sepharose affinity chromatography of trout post-heparin plasma resulted in a peak of LPL activity and no peak was obtained for SRL. The purified LPL showed high specific activity which was increased to several times than that of post-heparin plasma activity. 4.The effects of specific inhibitors on the purified enzyme was the same as obtained with trout post-heparin plasma. 5. The purified enzyme was shown to be serum dependent. A high stimulatory effect was obtained with trout serum VLDL, though stimulation was also obtained with HDL and LDL. The dependancy of the activity on serum gave evidence that the purified enzyme is serum stimulated lipase. iftory effects on the activity; this behaviour is. 6. The amounts of FAs liberated by the enzyme hydrolysis were proportioned to the incubation time. The rate of hydrolysis increased linearly with the amount of the enzyme. Increasing the amount of substrate resulted in a saturating of the enzyme, suggesting that. Michaelis-Mentlen kinetics were operative; an apparent of approximately Km was obtained. 7. Polyacrylamide gel electrophoresis of the purified enzyme showed only a single band. SDS polyacrylamide electrophoresis illustrated that the enzyme was composed of only one polypeptide with an apparent molecular weight of 63,000. 8. Different trout tissues showed low but variable activity; no activity was detected in some tissues but the highest activity was obtained in adipose tissue. 9. The highest activity from acetone-ether powder was by extraction with ammonia/HCl buffer containing sodium deoxycholate and potassium oleate. Heparin and glucose in the same buffer and also NaCl in sodium barbital buffer were less effective as extractants. 10. Adipose tissue activity was inhibited by the specific inhibitors, though little stimulation was obtained by heparin. Fresh tissue activity was stimulated by serum. These observations indicated that the enzyme in adipose tissue is similar to that of LPL in mammals. In the absence of serum acetone-ether powder, activity was increased by NaCl in a manner characteristic of SRL. 11. Heparin-Sepharose chromatography of adipose tissue produced two isolated activities, one was stimulated by NaCl and the other was inhibited by NaCl and stimulated by either serum or VLDL. 12. The presence of LPL in trout, with similar characteristics to those in mammals, strongly support the suggestion that fish transport and take up lipid by mechanisms analogous to those in mammals.