Paavo K. J. Kinnunen

An immunochemical method for the selective measurement of two triglyceride lipases in human postheparin plasma

A new method for the selective measurement of postheparin plasma lipoprotein lipase and hepatic lipase is described and validated. The activity of lipoprotein lipase is determined at 0.1 M NaCl after removal of hepatic lipase by specific antiserum, and the hepatic lipase is assayed in a medium containing 1.0 M NaCl but no additional serum. The optimal conditions for the determination of the two postheparin plasma triglyceride hydrolases were shown to be similar to those described for the purified enzymes. The new assay methods are simple, accurate and highly specific for the two lipase activities. VLDL and LDL do not interfere with the measurement, making the methods suitable for studies of patients with various hyperlipidemias. More than 90% of the total triglyceride hydrolase activity in postheparin plasma is precipitated with antisera raised against purified human postheparin plasma hepatic lipase and bovine milk lipoprotein lipase. The time and dose dependence of the two postheparin plasma lipase responses differ. For optimal activity of both enzymes, plasma taken 15 minutes after intravenous administration of 100 I.U./kg of heparin, should be used. The activity of postheparin plasma lipoprotein lipase and hepatic lipase in 12 young, healthy males is reported.

Vectorial budding of vesicles by asymmetrical enzymatic formation of ceramide in giant liposomes.

Sphingomyelin is an abundant component of eukaryotic membranes. A specific enzyme, sphingomyelinase can convert this lipid to ceramide, a central second messenger in cellular signaling for apoptosis (programmed cell death), differentiation, and senescence. We used microinjection and either Hoffman modulation contrast or fluorescence microscopy of giant liposomes composed of 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC), N-palmitoyl-sphingomyelin (C16:0-SM), and Bodipy-sphingomyelin as a fluorescent tracer (molar ratio 0.75:0.20:0.05, respectively) to observe changes in lipid lateral distribution and membrane morphology upon formation of ceramide. Notably, in addition to rapid domain formation (capping), vectorial budding of vesicles, i.e., endocytosis and shedding, can be induced by the asymmetrical sphingomyelinase-catalyzed generation of ceramide in either the outer or the inner leaflet, respectively, of giant phosphatidylcholine/sphingomyelin liposomes. These results are readily explained by 1) the lateral phase separation of ceramide enriched domains, 2) the area difference between the adjacent monolayers, 3) the negative spontaneous curvature, and 4) the augmented bending rigidity of the ceramide-containing domains, leading to membrane invagination and vesiculation of the bilayer.

Membranes: a meeting point for lipids, proteins and therapies

•  Introduction •  Membrane lipid composition •  Membrane lipid structure •  Membrane lipid organization ‐  Why so many different lipids? ‐  Lipid mixing and demixing ‐  Lateral pressure ‐  Surface electrostatics •  Role of lipids in cell functions •  Lipid influence in transmembrane protein function ‐  Prokaryotic potassium channel (KcsA) ‐  Mechanosensitive channels ‐  Voltage‐gated potassium channel (KvAP) ‐  Nicotinic acetylcholine receptor (nAcChR) ‐  G protein‐coupled receptors ‐  Other examples •  Non‐permanent proteins in membranes ‐  Proteins that interact reversibly with the bilayers ‐  Proteins that interact irreversibly with the bilayers ‐  Proteins that interact weakly with the membrane ‐  Proteins that interact strongly with the membrane ‐  G proteins and their interactions with membranes ‐  Small monomeric G proteins: the Ras and Ras‐like family ‐  Protein kinase C •  Membrane microdomains and lipid mediators in the control of heat‐shock protein response ‐  Stress sensing and signalling: the membrane sensor theory ‐  Hsp signalling in cancer and diabetes ‐  The role of membrane microdomains ‐  Lipid mediators of the stress response •  A subpopulation of Hsps can interact with and translocate through membranes ‐  Hsp90 in eukaryotic membranes ‐  Hsp70 in cell membranes ‐  Hsp27‐membrane interactions ‐  Secreted Hsps ‐  Representative cases where Hsps interact with membranes or release from the cells •  Concluding remarks

Increased plasma phospholipase-A2 activity in schizophrenic patients: Reduction after neuroleptic therapy ☆

Phospholipase-A2 (PLA2) is a key enzyme in the metabolism of phospholipids, and it may play an important role in neuronal function and neuronal plasticity. We determined the activity of PLA2 in the plasma of 20 drug-free schizophrenic patients, 6 nonschizophrenic psychiatric patients, and 21 healthy controls. Schizophrenic patients showed significantly higher plasma PLA2 activity than controls, and higher than our nonschizophrenic patients. Seventy percent of the schizophrenics had enzyme activity higher than the highest value from the control group. The increased plasma PLA2 activity in schizophrenics was reduced to the level of the controls after 3 weeks of neuroleptic treatment. These findings warrant further study for possible implications of this increased PLA2 activity in the etiopathology of schizophrenia.

Hepatic endothelial lipase antiserum influences rat plasma low and high density lipoproteins in vivo

Postheparin plasma contains two separate lipolytic enzymes, lipoprotein lipase and hepatic lipase, which are released from vascular endothelial cells. The former is located in extrahepatic capillary beds and has a well-defined role in the catabolism of plasma triglycerides. The hepatic lipase has been recently shown to be located on the surface of hepatic endothelial cells [l] but the physiological function of this enzyme is still far from clear. Hepatic endothelial lipase can hydrolyze chylomicron and VLDL triglycerides in vitro [2] but its activity in postheparin plasma has no correlation to plasma triglyceride levels [3]. It has been suggested that hepatic lipase could be involved in the uptake of chylomicron remnants [4], intermediate density lipoprotein [5] or LDL [6] by the liver but there is little experimental evidence for any of these possibilities. We have recently purified the heparin-releasable lipase from rat liver perfusates [7] and used the enzyme preparation for production of antiserum. The availability of this anti-hepatic lipase serum offered a good opportunity to study the function of the enzyme by searching whether plasma lipoproteins are influenced by specific inhibition of the hepatic endothehal lipase in vivo.

Molecular Mechanisms of Membrane Deformation by I-BAR Domain Proteins

BACKGROUND Generation of membrane curvature is critical for the formation of plasma membrane protrusions and invaginations and for shaping intracellular organelles. Among the central regulators of membrane dynamics are the BAR superfamily domains, which deform membranes into tubular structures. In contrast to the relatively well characterized BAR and F-BAR domains that promote the formation of plasma membrane invaginations, I-BAR domains induce plasma membrane protrusions through a poorly understood mechanism. RESULTS We show that I-BAR domains induce strong PI(4,5)P(2) clustering upon membrane binding, bend the membrane through electrostatic interactions, and remain dynamically associated with the inner leaflet of membrane tubules. Thus, I-BAR domains induce the formation of dynamic membrane protrusions to the opposite direction than do BAR and F-BAR domains. Strikingly, comparison of different I-BAR domains revealed that they deform PI(4,5)P(2)-rich membranes through distinct mechanisms. IRSp53 and IRTKS I-BARs bind membranes mainly through electrostatic interactions, whereas MIM and ABBA I-BARs additionally insert an amphipathic helix into the membrane bilayer, resulting in larger tubule diameter in vitro and more efficient filopodia formation in vivo. Furthermore, FRAP analysis revealed that whereas the mammalian I-BAR domains display dynamic association with filopodia, the C. elegans I-BAR domain forms relatively stable structures inside the plasma membrane protrusions. CONCLUSIONS These data define I-BAR domain as a functional member of the BAR domain superfamily and unravel the mechanisms by which I-BAR domains deform membranes to induce filopodia in cells. Furthermore, our work reveals unexpected divergence in the mechanisms by which evolutionarily distinct groups of I-BAR domains interact with PI(4,5)P(2)-rich membranes.

Effect of serum and C-apoproteins from very low density lipoproteins on human postheparin plasma hepatic lipase.

The presence of a lipase of hepatic origin in postheparin plasma of experimental animals has been confirmed by several investigators [1-6]. An enzyme with similar characteristics has also been found in humans [7-9]. This enzyme hydrolyzes triglycerides, phospholipids and acylCoA thiolesters [4,9] and has been shown to be a transacylase [10]. Hepatic lipase clearly differs from lipoprotein lipase of extrahepatic origin in its co-factor, requirement and immunological reactivity. Also, it is not inhibited by high ionic strength in the assay medium [5,7,11,12]. One of the characteristics of lipoprotein lipase is that it needs for full activity a VLDL-apoprotein, apoC-II. This has been demonstrated for purified lipoprotein lipase from adipose tissue [14], heart [15], postheparin plasma [4] and milk [16]. A recent study with bovine milk lipoprotein lipase indicates, that depending on the substrate concentration and the presence of apoC-lI in the assay medium, also apoC-I and apoC-III can stimulate fatty acid liberation [ 17]. We now report, that low concentrations of serum activate purified human postheparin plasma hepatic lipase. The C-apoproteins from VLDL did not cause this stimulation, but all of them suppressed the enzymatic activity. 2. Material and methods

Lipid dynamics and peripheral interactions of proteins with membrane surfaces

A large body of evidence strongly indicates biomembranes to be organized into compositionally and functionally specialized domains, supramolecular assemblies, existing on different time and length scales. For these domains and intimate coupling between their chemical composition, physical state, organization, and functions has been postulated. One important constituent of biomembranes are peripheral proteins whose activity can be controlled by non-covalent binding to lipids. Importantly, the physical chemistry of the lipid interface allows for a rapid and reversible control of peripheral interactions. In this review examples are provided on how membrane lipid (i) composition (i.e., specific lipid structures), (ii) organization, and (iii) physical state can each regulate peripheral binding of proteins to the lipid surface. In addition, a novel and efficient mechanism for the control of the lipid surface association of peripheral proteins by [Ca2+], lipid composition, and phase state is proposed. The phase state is, in turn, also dependent on factors such as temperature, lateral packing, presence of ions, metabolites and drugs. Confining reactions to interfaces allows for facile and cooperative large scale integration and control of metabolic pathways due to mechanisms which are not possible in bulk systems.

Effect of Oxandrolone Treatment on the Activity of Lipoprotein Lipase, Hepatic Lipase and Phospholipase a1 of Human Postheparin Plasma

The effect of a synthetic steroid, oxandrolone, on total postheparin plasma lipolytic activity, postherpain hepatic lipase activity, lipoprotein lipase and phospholipase A1 was studied in seven patients with hypertriglyceridemia. The mean total postheparin lipolytic activity increased 100 per cent during oxandrolone tratement (p smaller than 0.05). This change was caused mainly by postheparin hepatic lipase, whose activity increased on the average more than 2.5 times (p smaller than 0.001). The change in postheparin plasma-lipoprotein-lipase activity was insignificant. A highly significant correlation (r equals +0.87, p smaller than 0.01) was observed between the activities of postheparin hepatic lipase and phospholipase A1 before and during oxandrolone treatment. No relation was observed between serum triglyceride level and various postheparin lipase activities, or between the changes induced by oxandrolone in the level of serum lipids and the activities of postheparin lipases. We conclude that oxandrolone increases the activities of postheparin plasma hepatic lipase and phospholipase A1 but has little influence on lipoprotein lipase.

Lipid microdomains in dimyristoylphosphatidylcholine-ceramide liposomes.

Binary membranes composed of dimyristoylphosphatidylcholine (DMPC) and natural ceramide (up to a mole fraction Xcer = 0.25) were investigated by measuring the excimer:monomer fluorescence emission intensity ratio IE:IM for the pyrene labeled phospholipid probe 1-palmitoyl-2[(pyren-1-yl)]decanoyl-sn-glycero-3-phosphocholine (PPDPC), by monitoring fluorescence polarization of diphenylhexatriene (DPH), as well as using differential scanning calorimetry (DSC). Increasing Xcer > 0.10 both below and above the main transition temperature Tm increased IE:IM for PPDPC maximally approximately 1.6-fold at Xcer = 0.25. Above Tm, and when Xcer approaches 0.10, fluorescence polarization P for DPH increases steeply, reflecting an overall decrease in acyl chain motions. At Xcer = 0.10 there is a discontinuity in P and upon further increase in the content of ceramide a smaller, yet significant increase in P is evident. DSC revealed ceramide to increase in the pretransition temperature until at Xcer exceeding 0.07 this transition was no longer evident. Simultaneously, increasing Xcer up to 0.05 increased Tm from 23.9 to 24.6 degrees C. Total enthalpy delta Hm of the main transition diminished progressively upon increase in Xcer up to approximately 0.10. Above this concentration of ceramide a new endotherm became evident at 22.5 degrees C, and exceeding Xcer = 0.14 this endotherm became dominant. Our results indicate an enrichment of the pyrene labeled phospholipid analog into microdomains concomitant with the formation of a distinct ceramide-enriched phase at Xcer > 0.10.