Febs Letters Volume 53, Number 1-3./Volume 54, Number 1-3. [antikvár]

Volume 53, number 1 FEBS LETTERS April 1975 ISOLATION OF C-I AND C-II ACTIVATED LIPOPROTEIN LIPASES AND PROTAMINE INSENSITIVE TRIGLYCERIDE LIPASE BY HEPARIN-SEPHAROSE AFFINITY CHROMATOGRAPHY Devaki GANESAN and Helen B. BASS Cardiovascular Research Program, Oklahoma Medical Research Foundation, and the Departments of Medicine and Biochemistry and Molecular Biology, Oklahoma City, Oklahoma 73104, USA. Received 12 March 1975 1. Introduction 2. Materials and methods Recent evidence from several laboratories has indicated the presence of at least three long chain triglyceride hydrolases in the post-heparin plasma of normoli-pidemic subjects and laboratory animals [1—7]. These triglyceride hydrolases have the following properties: 1) Lipoprotein hpase C-1 (LPI^j) is present in post-heparin plasma but not in adipose tissue from normal controls or post-heparin plasma from subjects with Type 1 hyperlipoproteinemia. LPLf ] is activated by serum or C-1 (one of the apoUpoprotein polypeptides of lipoprotein C) but not C-II and is inhibited by NaCl and protamine sulfate [7]. 2) Lipoprotein hpase C-Il (LPLq.jj) is present in post-heparin plasma and adipose tissue [7-10]. LPL^.u is activated by serum or C-Il but not C-I and is inhibited by NaCl and protamine sulfate. 3) A third triglyceride lipase (TGL) differs from the foregoing two lipases in that it is insensitive to inhibition by protamine sulfate and 1 M NaCl [1,2]. TGL is believed to be secreted from liver [5] and is not activated by serum or apolipoprotein cofactors [15]; LPL^.u [6] and TGL [4-6], but not LPLc.i have been isolated by heparin-sepharose chromatography. The present report describes the isolation of all three Hpases by heparin-sepharose chromatography of post-heparin plasma from normal controls, and demonstrates that the 1.5 M NaCl eluate which contains LPL^,.,, [4-6] also exhibits LPL^.i activity following desalting of this fraction. 2.1. Source of post-heparin plasma Post-heparin plasma was obtained from healthy donors who had fasted overnight (14—16hr). Blood was drawn into iced plastic bags containing 1/50 vol of 4% sodium citrate 30 min after intravenous administration of 10 000 units of heparin sodium (Upjohn Co., Kalamazoo, Michigan). Plasma was separated by centrifugation (4,500 rev/min) at 0°C and stored at -15°C. 2.2. Isolation of LPLq_j and LFLq_h by Sephadex G-lOO chromatography A preparation containing LPLf-.j and LPI.(;.|j, which had been isolated as described previously [11], was applied to a column (1.5 X 100 cm) containing Sephadex G-lOO equUibrated with 0.05 M NH4OH-NH4CI buffer, pH 8.5. Additional buffer (4''C) was applied at the rate of 20-25 ml/hr and the eluate was collected in 2.0-ml fractions. Each fraction was analyzed for protein [12] and lipolytic activity using ['''C] -triolein activated by serum, C-I or C-Il. 2.3. Isolation ofLPL^.j, LPLc-n and TGL by heparin-Sepharose chromatography Heparin-Sepharose column chromatography was performed according to the procedure described by Böberg, et al. [6]. Small columns (0.4 X 10 cm) were filled to a height of 2.5 cm with heparin-Sepharose, prepared according to the method of Egelrud and North-Holland Publishing Company - Amsterdam 1