Cell Membranes

Membranes are barriers for hydrophilic molecules and ions because of the hydrophobic core of the phospholipid bilayer. A membrane or phospholipid bilayer is a two-dimensional, spherical particle separating an inside compartment from an outside compartment. In addition to lipids, a membrane contains membrane proteins that control the transport of hydrophilic and charged, small and large molecules into and out of the cell and intracellular organelles. The importance of the lipid bilayer membrane is its ability to function as an electrical capacitor. This enables charge separation and thus the storage of electro-chemical energy in form of ion gradients. One example is the proton motif force (pmf or proton gradient) discussed in sections on oxidative phosphorylation and photosynthesis. Membrane proteins that serve as conductors are used by the cell to extract small quanta of this energy for synthesis or signaling mechanisms. Two examples are ATP synthesis and action potentials, respectively.

Phospholipids
(KEGG pathways MAP00561)

The main structural component of biological membranes are the phospholipids. The biosynthesis phospholipids depends on the syntesis of diacylglycerol. Diacylglycerol in liver comes from phosphatidic acid (C00416; 1,2-Diacyl-sn-glycerol 3-phosphate). Phosphatidic acid can be derived from glycerol (C00116) by phosporylating it at the expense of ATP to glycerol-3-phosphate (C00093). The enzyme catalyzing the reaction is glycerol kinase (EC 2.7.1.30). Glycerol-3-P can also be obtained from reduction of the glycolytic intermediate dihydroxyacetonephosphate (DAHP; C00111) by glycerol-3-P dehydrogenase (EC 1.1.99.5,  1.1.1.94, 1.1.1.8 isoforms). Glycerol-3-phosphate is then twice esterified using acyl-CoA precursors as fatty acid donors to phosphatidic acid. The first acylation to 1-Acyl-sn-glycerol 3-phosphate (C00681) is catalyzed by Glycerol-3-phosphate O-acyltransferase (EC 2.3.1.15) and the second fatty acylation is catalyzed by 1-Acylglycerol-3-phosphate O-acyltransferase (EC 2.3.1.51) to 1,2-Diacyl-sn-glycerol 3-phosphate (systematic name) or phosphatidic acid (common name).

The first acylation of glycerol-phosphate takes place at either the endoplasmatic reticulum membrane or the mitochondrial membrane. The latter reaction is significant in liver, an organ with a large numbers of mitochondria. The product of this acylation is lysophosphatidic acid. Note that at this time this and all following metabolites are membrane components, including all enzymes catalyzing the synthesis of membrane lipids.

Phosphatidic acid is a minor component of cell membranes but an essential intermediate for both fat synthesis and phospholipid synthesis. Fat or triglyceride (C00422) are synthesized from phosphatidic acid by dephosphorylation to 1,2-diglyceride (C00641) by Phosphatidate phosphatase (EC 3.1.3.4). A third acyl-CoA unit is then used to esterify the third carbon position in the glycerol backbone by the enzyme Diglyceride acyltransferase (EC 2.3.1.20) to form triglycerides.

1,2-diglyceride is a precursor for both phosphatidylcholine (C00157, lecithin) and phosphatidylethanolamine (C00350) by combining the diacylglycerol with an activated choline (ethanolamine) group using the nucleoside CTP to form CDP-choline (C00307) and CDP-ethanolamine (C00570), respectively. Choline is activated by phosphorylation using ATP to phosphorylcholine and phosphorylcholine in turn is formed to CDP-choline by CTP releasing pyrophosphate as byproduct.

These committed reactions are analogous to the activation of glucose-1-P to UDP-glucose for glycogen synthesis. Phosphatidylethanolamine can also serve as precursor for phosphatidylcholine by transfer of three activated choline groups In addition, phosphatidylserine is derived from phosphatidylethanolamine by exchanging the ethanolamine headgroup for serine.

Two minor phospholipids are phosphatidylglycerol (PG) and phosphatidylinositol (PI). They are derived from phosphatidic acid instead of 1,2-diglyceride. Phosphatidic acid is activated by activation using CTP to form the high energy compound CDP-diglyceride (C00269). The cytosine unit is replaced by either inositol to form phosphatidylinositol (PI) and CMP, or glycerol-3-phosphate to form phosphatidylglycerol (PG) and CMP and P_i. Both PG and PI are negatively charged phospholipids because of the single negative charge contributed by the phosphate ester linkage.

Fatty acid elongation and desaturation (oxidation)

Phospholipids can be distinguished not only by their headgroup chemical properties, but also their fatty acid composition. The latter varies in terms of acyl chain length and degree of desaturation, or oxidation. The cytoplasmic fatty acid synthetase complex only forms palmitoyl-CoA as end product. Longer fatty acids, predominantly C18 and the monounsaturated derivatives of C16 and C18 are commonly found in biological membranes.

In eukaryotes the elongation of of fatty acids beyond C16 length is performed by enzyme systems of the endoplasmatic reticulum membrane, with the active sites facing the cytoplasmic side of this membrane. The substrate for fatty acid elongation and desaturation is the acyl-CoA form, although some cells or organisms may use directly phospholipids. The diverse acyl-CoA moieties are subsequently acylated to glycerol-3-P and lysophosphatidic acid as described above.

To elongate fatty acyl chains (C16 or longer) by C2 units at their carboxyl end, malonyl-CoA is used as donor for both saturated and unsaturated fatty acids. The elongation process starting from C16:0 can be repeated to produce C18:0, C20:0, C22:0, and C24:0 fatty acids. These latter fatty acids are important for sphingomyelin formation in brain.

Oxidation (desaturation) requires NADPH/H⁺ and molecular oxygen in a reaction involving a three-enzyme complex: NADPH-cytochrome b5 reductase (FAD coenzyme), cytochrome b5 (heme), and D9-desaturase (non-heme iron). The latter oxidizes stearoyl-CoA to oleoyl-CoA. The reducing equivalent from NADPH/H⁺ together with the electron/hydrogen pairs gained from cis C=C bond formation are used to reduce one molecular oxygen to two water molecules. The net oxidation of the saturated C18:0 fatty acid (stearoyl) to a monounsaturated state (C18:1; oleoyl) is given as:

                    Stearoyl-CoA + NADPH + H⁺ + O₂ Þ Oleoyl-CoA + NADP+ + 2H₂O

A single cis C=C bond is introduced in this reaction resides between carbon 9 and carbon 10. The bond is referred to as cisD9. As an example, look at Phosphatidylcholine desaturase (EC 1.3.1.35). Its substrates are linoleate and oleate.

Note, there is no pathway included in KEGG on phospholipid elongation or desaturation. Desaturases of fatty acids do not produce chemical energy and their mechanism is fundamentally different from the dehydrogenase activities of mitochondrial enzymes of the Krebs cycle and beta-oxidation pathways. Here, reducing power is invested to support the oxidation of fatty acids. These desaturases are also distinct from so called monooxygenases (cytochrome P450 or mixed function oxygenases) which will be discussed in the chapter on sterol synthesis. As their name suggests, the lipid substrate is oxygenated (epoxide, hydroxyl) in the process.

Mammals do not have enzymes to oxidize fatty acids carbon bonds beyond the C9-C10 position. Thus in mammals there exist desaturases for positions 4, 5, and 6, but not for 12 or 15. Most polyunsaturated fatty acids in human metabolism depend on the two essential fatty acids. Linoleate (C18:2 D9,D12; KEGG C01595) and linolenate (C18:3 D9,D12,D15) are part of dietary lipids and are precursors in mammalian cells for the synthesis of eicosanoids that function as signaling molecules in cell-cell communication, development, and inflammation (prostaglandin and leukotrienes).

Assembly of lipids into membranes

The process of lipid insertion into membranes can be split into three major parts. First, the initial incorporation of newly synthesized lipid into an acceptor membrane. Second, the generation of the proper, i.e., asymmetric, transmembrane distribution of lipids. Third, maintenance of the proper lipid composition in the various target membranes for which de novo synthesized lipids have been produced. The mechanism of the first step appears obvious based on the above discussed ER membrane bound enzyme systems responsible for the synthesis of phosphatidic acid, phospholipids, and triglycerides. The substrates and products of these enzyme mechanisms are located in the outer leaflet of the ER membrane. The second mechanism requires the redistribution of phospholipids from the outer to the inner leaflet (or monolayer) of the ER membrane. This process is not random, for all cell membranes are found to contain an asymmetric composition of lipid components in the inner and outer monolayers. Specialized membrane proteins called flippases transport the hydrophilic headgroup portion of these amphipathic molecules across the hydrophobic fatty acid core of the ER membrane.

Little is known about the specifics of these transport and sorting mechanisms. One membrane protein in ER membranes is an ATP driven 'pump' that shuttles phospholipids from the cytoplasmic monolayer, the site of synthesis, to the lumenal side, which corresponds to the extra-cellular side of the plasma membrane. An alternative mechanism has been suggested for the synthesis of PC, which is found preferentially on the cell surface. In this model, a transmembrane methylase uses PE on the cytoplasmic side of the membrane as substrate and sequentially methylates the headgroup to choline forming PC. In the process, the phospholipid is transported across the membrane with the choline headgroup facing the lumenal side. De novo synthesized membranes are transported via a vesicle mechanism to the Golgi complex and from there to different organelles and the plasma membrane. The orientation of the two monolayers, the cytoplasmic surface and the lumenal side, are strictly maintained.

Lipoprotein particles

The major site of lipid synthesis is the endoplasmatic reticulum membrane of the liver. Lipids therefore have to be transported to other membranes in the same cell and from liver cells to other tissues. Inside cells, membrane vesicles are transported between various organelles and the plasma membrane. Outside cells, lipids are transported in association with serum proteins, albumins and lipoprotein particles.

Lipoprotein particles are heterogeneous complexes with various blood plasma protein fractions carrying triglycerides, phospholipids and cholesterol. Together, they compose the blood lipid levels which measures about 500mg/100ml and exhibits wide fluctuations depending on dietary sources. Lipoprotein particles are distinguished on the basis of their physical density. Chylomicrons show the lowest density of all lipoproteins having the highest lipid to protein ratio of all. Chylomicrons are composed mostly of fats (from diet absorption) with a thin protein layer that keeps the particle soluble. The very low density lipoproteins (VLDL) have a high lipid content but contain more cholesterol and phospholipids making them more dense than chylomicrons. Low density lipoproteins (LDL) have the highest ratio of cholesterol, while high density lipoproteins (HDL) have the highest phospholipid content. The very high density lipoproteins (VHDL) contain mostly albumin and carry mostly free fatty acids.

With the exception of albumin, all protein components of lipoprotein particles are classified as globins. Alpha globins are the major component of HDLs and beta globins the major component of LDLs. Each of the lipoprotein classes play a distinct role in lipid transport. VLDLs carry fats from liver to other tissues (dietary fats come from mucosal cells via chylomicrons). The major target tissue is adipose tissue. After unloading its triglycerides the VLDL is converted to an LDL particle. LDL carries the bulk of plasma cholesterol. The targets are peripheral tissues and the cholesterol exists in an esterified form. The origin of these cholesterol esters is liver cholesterol which upon its secretion from hepatocytes is acylated by the plasma enzyme lecithin-cholesterol acyl transferase that transfers an unsaturated fatty acid from position 2 of phosphatidylcholine to cholesterol. The left-over phospholipid - lysophosphatidylcholine- can be re-acylated in red blood cells to PC. The exchange of lipids from lipoproteins occurs directly with the cell membranes of the target cells. The unloaded lipoprotein is now a VHDL containing mostly protein fractions (albumin bound fatty acids) and can be recycled by the liver.

Atherosclerosis

Atherosclerotic lesions consist of cholesterol deposits in the intimal lining of the arteries. Eventually, the lumen may become occluded to the extent that the blood flow is impaired. This causes restricted oxygen supply to peripheral organs like the brain, back pressure on the major arteries that can lead to high blood pressure and congestive heart failure. Blood platelets and debris can accumulate at the plaque deposits further narrowing the circulation. Chronic high ratios of LDL ('bad cholesterol') over HDL particles is thought to be the major cause of these cholesterol deposits.

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