1. PART 2 BIOCHEMICAL REGULATION DR SAMEER FATANI BIOCHEMISTRY

2. Plasma membrane structure and metabolite transport Structure and membrane transport Introduction: Biological membranes from eukaryotic and prokaryotic cells have many properties in common such as: same chemical components and similar structural organization. The major differences could be in specific lipids, protein and carbohydrate components. The mammalian membranes have trilaminar appearance with overall width of 7-10 nm. Intracellular membranes are usually thinner than plasma membrane.

3. Functions of the membranes: Membranes are (very dynamic structures) usually undergo specific movement to give the cells and subcellular structures the ability to adjust their shape and change the position. However, the chemical components, proteins and lipids are involved in this dynamic role. The cellular membranes can control the composition of the cells by: -preventing a variety of molecules to enter the cells - allowing the movement of specific molecules from one side to another by selective transport systems.

4. Chemical composition of the membranes: Lipids and proteins are the two major components of all membranes. The amount of each varies greatly in different membranes (12.2). Protein ranges from about 20% (as in myelin sheath ) to over 70% (as in inner membrane of mitochondria). Intracellular membranes have a high percentage of protein due to the large number of enzymatic activities available. Membranes contain small amount various polysaccharide in the form of glycoprotein and glycolipid, but contain no free carbohydrate.

5. MEMBRANE LIPIDS: There are three major types of lipids in biological membranes: 1- Glycerophospholipids: are the most abundant lipids of membranes. Two types of basic structure: Basic structure number 1: Phosphatidic acid : where different hydroxyl containing compounds (such as: choline, ethanolamine, or serine) are esterified to its phosphate group to form glycerophospholipids.

6. Phosphate group Glycerol molecule Two long chain fatty acids Sat. FA at C1 & Unsat. FA at C2 Any hydroxyl containing compound: e.g. Choline, Ethanolamine, Serine, & Inositol Phosphatidic acid Esterification Esterification at C1 & C2 of gly. Esterification Glycerophospholip id Basic structure for glycerophospholipids

7. the most common glycerophospholipids: - Ethanolamine glycerophospholpid or cephalin - Choline glycerophospholipid or Lecithin -Diphosphatidylglycerol or Cardiolipin inner mitochondrial membrane in most membranes Another basic structure for glycerophospholipids Plasmalogen (Glycerol Ether phospholipids) : contain an alkyl group or unsaturated ether instead of fatty acid in C1 Of glycerol molecule Glycerophospholipids are amphipathic, contain both: -Polar end or head, due to the charged phosphate and subsitutions on phosphate -Non polar tail, due to hydrophobic hydrocarbon chains of fatty acyl groups.

8. 2- Sphingolipids: amino alcohols. The basic structure: sphingosine and dihydrosphingosine e.g. Ceramide has Unsat. F.A. in amide linkage on the amino group e.g. sphingomyelin has phosphorylcholine esterified to the 1-OH, (abundant in mammalian tissues) - Glycossphingolipids: no phosphate group, contain a sugar molecule Attached by glycosidic linkage to the C1 of sphingosine. e. g. Ganglioside: most complex glycosphingolipids, contain Oligosaccharide with one or more residues of sialic acid 3- Cholesterol: is an important component of plasma Membranes. - Composed of four fused rings and branched hydrocarbon chain (C8) attached to D rings. (fig. 12.16—D5). - Compact, rigid, and hydrophobic molecule. -It has a polar hydroxy group (HO) at C3 - cellular function: cholesterol can alter the fluidity of membranes and participates in controlling the microstructure of plasma membranes.

9. the amount of lipids in cell membranes: -Plasma membrane has the greatest variation in percentage of lipid composition, because the quantity of cholesterol can be affected by the nutritional state of animal. -The same intra cellular membranes of certain tissue in different species have very similar classes of lipids e.g. Myelin membranes: rich in sphingolipids with high glycosphingolipids, while intracellular membranes primarily contain glycerophospholipids and little sphingolipids -The constancy of composition of different membranes indicates the relationship between lipids and the specific functions of individual membranes.

10. MEMBRANE BROTEINS -Peripheral proteins : located on the surface of membrane. E.g. electrostatin (electrostatic binding) -Integral proteins (IP): Two types: 1- proteolipids: hydrophobic lipoproteins. - soluble in chloroform and methanol but not water). - present in many membranes, particularly myelin (50% or protein component) - e.g. lipophilin (in brain myelin). 2- Glycoproteins: contain carbohydrates, present mainly in plasma membranes. e.g. glycophoryn. IP contain bound lipids (if removed lead to Denaturation of proteins and loss of biological functions). It may also contain hydrophobic Amino acids.

11. functions of membrane proteins: - Have a role in transmembrane movement of molecules - Act as receptors for binding hormones and growth factors -In many cells (e.g. neurons) membrane proteins have structural role to maintain the shape of the cell. MEMBRANE CARBOHYDRATES In membranes, carbohydrates are present as oligosaccharides attached covalently : -to proteins to form glycoproteins -or to lipids to form glycolipids Their sugar include: glucose, galactose, mannose, N- acetylgalactosamine, N-acetylglucsamine, and sailic acid. Carbohydrate is located on the exterior side of plasma membrane or luminal side of the endoplasmic reticulum. Functions of plasma carbohydrates: -cell-cell recognition -Adhesion - And receptor action

12. STRUCTURE OF BIOLOGICAL MEMBRANES Fluid Mosaic Model of Biological Membranes: Introduction. All biological membranes have a bilayer arrangement of lipids. Amphipathic lipids and cholesterol are oriented so that their hydrophobic portions interact to minimize their contact with water or polar groups. (fig. 12.22--- D5). Fluid mosaic model for membranes in which: -Some proteins (intrinsic) are actually immersed in the lipid bilayer. While others (extrinsic) are loosely attached to the surface. -therefore, it was suggested that some proteins span the lipid bilayer and are in contact with the aqueous environment on both sides. Fig. 12.22--- D5). The characteristics of lipid bilayer has a relationship with the properties of the cellular membranes, including: fluidity, flexibility that permits changes of shape and form, ability to self heal and impermeability.

13. Asymmetric distribution of lipid Lipid components are distributed in asymmetric manner across the biological membranes. Each layer of the bilayer has different composition with respect to individual glycerophospholipids and sphingolipids. E.g. the asymmetric distribution of lipids in human erythrocyte membrane: Sphingomyelin is predomenantly in outer layer, whereas phosphatidyl ethanolamine is predomenantly in the inner layer, in contrast cholesterol is equally distributed on both layers. Asymmetry of lipids is maintained by specific membrane proteins, termed lipid transporters: -Flipase: catalyzes the inward transport -Flopase: catalyzes the outward transport -Scramblase: mixes phospholipids between the two layers.

14. Movement of molecules through membranes The lipid nature of biological membranes strongly restricts the type of molecules that diffuse from one side to another. Inorganic ions and charged organic molecules do not diffuse at a significant rate because of their attraction to water molecules And also due to the hydrophobic environment of Lipid membranes. The size of large molecules such as proteins and nucleic acids preclude significant diffusion. To overcome this and to transport such molecules across membranes, a variety of specialized channels and transporters are involved. Mediated transport system (facilitated): Membrane channels: membranes of most cells contain specific channels (pores), these channels permit rapid movement of Specific molecules or ions from one side of a membrane to the other. The substances can diffuse in both directions of the membrane Via an aqueous holes. Channel protein do not bind the molecules or ions to be transported. The channels have some degree of specificity, based on size and charge of substance.

15. Transporters: the transporters translocate the molecule or ion across the membrane by binding to physically moving the substance (no chemical reactions occurs). Types of transporters: 1- passive transport (facilitated diffusion): the transporters move the substrate only down their concentration gradient. 2- Active transport: transporters move the substrate against it’s concentration gradient, and require some form of energy. With both channels and transporters the molecule is unchanged after translocation across the membrane. A major difference between channels and transporters is the rate of substrate translocation. Group translocation: involves not only movement of a substance across a membrane but also a chemical modification of the substrate during the process. e.g. Uptake of sugars by bacteria involves transport and then phosphorylation of the sugar before release into the cytosol.

16. Characteristics of membrane transport systems: The proteins or protein complexes involved in transport systems have a number of characteristics (fig. 12.5--- D5): -They facilitate the movement of a molecule or molecules through the lipid bilayer at a rate that is significantly faster than can be accounted by simple diffusion. -As the concentration of the substance to be translocated increases, the initial rate of transport increases but reaches maximum when the substance saturates the protein transporter. Simple diffusion does not demonstrate saturation kinetics. - most transporters have structural and stereo specificity for the substance transported. -Structural analogs of the substrate inhibit competitively and reagents that react with specific groups on proteins are noncompetitive inhibitors.

17. Four common steps in the transport of solute molecules (fig. 12.32---- D5) 1 - recognition by the transporter f appropriate solute from a variety of solutes in the aqueous environment. 2- translocation of solute across the membrane. 3- release of solute by the transporter, and 4- recovery of transporter to its original condition to accept Another solute molecule. The above four steps for the movement of single solute Molecule by a transporter. There are systems that move two Molecules simultaneously in one direction, called (symport mechanisms). Two molecules in opposite directions, (antiport mechanism). Single molecule in one direction (uniport mechanism).

18. Energetics of membrane transport systems the change in free energy when an unchanged molecules Moves from concentration C1 to concentration C2 on the other side of a membrane is given by the following Eq. ΔG’ = 2.3RT log (C2/C1) A facilitated transport system is one in which ΔG’ is negative and the movement of solute occurs spontaneously, without the need for a driving force. When ΔG’ is positive, as would be the case if C2 is larger than C1, coupled input of energy from some source is required for movement of the solute and the process is called active transport. Active transport is driven by either hydrolysis of ATP to ADP or utilization of an electrochemical gradient of Na + or H + across the membrane.

19. CHANNELS AND PORES Channels and pores in membranes function differentialy: Channels and pores are intrinsic membrane proteins and are differentiated on the basis of their degree of specificity for Molecules crossing the membrane. - Channels are selective for specific inorganic cations and anions, -Whereas pores are not selective, permitting organic and inorganic molecules to pass through the membrane. e.g. Na+ channel permits movement of Na+ at rate ten times greater than K+. The mechanisms of opening and closing the channels: Opening and closing of membrane channels involves a conformational change in the channel protein, in turn this conformational change is controlled by the transmembrane Potential (these channels called voltage-gated channels). e.g. in Na+ channel, depolarization of the membrane lead to an opening of the channel. Binding of specific agent, termed an agonist, is another Mechanism to control opening of a channel. e.g. binding of acetylcholine opens the channel in The nicotinic-acetylcholine receptor.

20. Sodium channel Voltage-sensetive Na+ channels mediate rapid increase In intracellular Na+ following depolarization of the plasma Membrane in nerve and muscle cells. There are four repeat homology units in the channel. Each With six transmembrane α helices. One membrane segment has a positively charged amino acid at every third position and may serve as a voltage sensor. A mechanical shift of this region due to a change in the membrane potential may lead to a conformational change in the protein, resulting In the opening of the channel. Nicotinic-Acetylcholine channel (nAChR) The nicotinic-acetylcholine channel (acetylcholine receptor), is An example of a chemically regulated channel, in which binding Of acetylcholine opens the channel and allowing selective Cations to move across the membrane. the nicotinic-acetylcholine receptor is inhibited by several deadly Neurotoxins including d-tubocurarine.

21. PASSIVE MEDIATED TRANSPORT SYSTEMS Passive mediated transport (facilitated diffusion) is a mechanism for translocation of solutes through cell membranes (from higher to lower concentration) without expenditure of metabolic energy. Glucose transport is facilitated: Eight members of superfamily of membrane proteins that mediate D-glucose transport have been reported in mammalian cells, and are expressed in a tissue-specific manner. The glucose transporters are designated as GLUT1, GLUT2, and so on. All have 12 hydrophobic segments considered to be the transmembrane regions. Most are in the plasma membrane where direction of movement of glucose is usually out to in. GLUT2, however, may be responsible for glucose export from liver cells. GLUT5 of sarcolemmal membranes of skeletal muscle transport fructose preferentially. GLUT4 is an insulin-responsive transporter. Several sugar analogs as well as phoretin are competitive inhibitors.

22. Cl- and HCO+ transport system An anion transporter in erythrocytes and kidneys involves the antiport movement of Cl- and HCO3-. This transporter called “the Na+-independent Cl- - HCO- exchanger”. The direction of the flow is reversible and depends on the concentration gradients of the ions across the membrane. The transporter is important in adjusting the erythrocyte HCO3- concentration in arterial and venous blood. Mitochondria contain a number of transport systems: The inner mitochondrial membrane contains antiport systems for exchange of anions between cytosol and mitochondrial matrix, including: 1- a transporter for exchange of ADP and ATP, 2- a symport transporter for phosphate and H+, 3- a dicarboxylate carrier that catalyzes an exchange of malate for phosphate, and 4- a translocator for exchange of aspartate and glutamate. These transporters mediate passive exchange of metabolites down their concentration gradient.

23. ACTIVE MEDIATED TRANSPORT SYSTEMS Characteristics: - they require utilization of energy to translocate solutes. - Saturation kinetics, specificity, and inhibatory. Classification : 1- Primary active transporters : if they require ATP as the energy source. Types of primary active transport: - P type transporters ( if he protein phosphorylated and dephosphorylated during the transport activity). -V vacuole type: responsible for acidification (proton pumps) of the interior of lysosomes, endosomes…….. - F type transporters: are involved in ATP synthesis. 2- Secondary active transporters : if a transmembrane chemical gradient of Na+ or H+ is required for translocation of sugars and amino acids. Translocation of Na+ and K+ is by primary active transport: All mammalian cells contain a Na+ - K+ transporter, of type P, which utilizes ATP to drive the translocation.

24. Na+/K+ - exchanging ATPase In all plasma membranes: the enzyme ATPase will be activated and catalyzes the reaction: ATP Na+ + K+ ADP + Pi Mg2+ -The function of this enzyme and reaction is to translocate Na+ and K+ across the biological membrane (3 Na+ ions moving out and 2 K+ ions into the cell). The enzyme has high activities in excitable tissues such as muscle and nerves. -Antiport process

25. Sodium/ glucose cotransporter (Secondary active transport systems) Transport of D-glucose is driven by movement of Na+. The Na+ electrochemical gradient across the plasma membrane Is an energy source for active symport (moving two molecules in One direction) movement with Na+ of sugars, amino acids, and Ca+. In the process, two Na+ are moving by passive facilitated transport Down the electrochemical gradient and glucose in the same time will Be carried along against its concentration gradient. Na+ gradient is maintained by the Na+/K+ -exchange ATPase Amino acids are translocated by luminal epithelial cells of intestines by Na+/amino acid cotransporters by a symport mechanism. Symport movement of molecule utilizing the Na+ gradient involves Cooperative interaction of Na+ ions and another molecule translocated On the protein. A conformational change of the protein occurs following Association of both ligands. Which moves them the necessary distance In the transporter to bring them into contact with cytosolic environment. Dissociation of Na+ ion from the transporter because of the low intracellular Na+ concentration leads to a return of the protein to its original conformation And a decrease in affinity and release of the other ligand (fig. 12.51----D5)

26. IONOPHORES It’s a class of antibiotics of bacterial origin that facilitate the Movement of monovalent and divalent inorganic ions across The biological and synthetic membranes. Two major groups: - Mobile carriers ionophores: can diffuse in the biological membrane and carry an ion across a membrane. - Channel formers ionophores: can create a channel that transverses The membrane and through which ions can diffuse e.g. Valinomycin transport K+ by an electrogenic uniport mechanism that creates an electrochemical gradient across a membrane as it carries a positive charged K+.