Biochemistry is the language of biology. The tools for research in all the branches of medical science are mainly biochemical in nature. The study of biochemistry is essential to understand basic functions of the body. How the food that we eat is digested, absorbed, and used to make ingredients of the body? How does the body derive energy for the normal day to day work? How are the various metabolic processes interrelated? What is the function of genes? Answer for such basic questions can only be derived by a systematic study of biochemistry.
Modern day medical practice is highly dependent on the laboratory analysis of body fluids, especially the blood. The disease manifestations are reflected in the composition of blood and other tissues. The study of biochemistry is necessary to give the scientific basis for disease and is useful for intelligent treatment of patients.
The practice of medicine is both an art and a science. The word "doctor" is derived from the Latin root, "docere", which means "to teach". The word chemistry is derived from the Greek word "chemi" (the black land), the ancient name of Egypt, when science was called the "black art". Indian medical science, even from ancient times, had identified the metabolic and genetic basis of diseases. Charaka, the great master of Indian Medicine, in his treatise (circa 400 BC) observed that madhumeha (diabetes mellitus) is produced by the alterations in the metabolisms of carbohydrates and fats; the statement still holds good.
The term "Biochemistry" was coined by Neuberg in 1903 from Greek words, bios (= life) and chymos (= juice). Some of the important milestones in the development of science of biochemistry are given in Table 1.1. Biochemistry is the most rapidly developing subject in medicine. Thanks to the advent of DNA-recombination technology, genes can now be transferred from one person to another, so that many of the genetically determined diseases are now amenable to gene therapy.
More than 99% of the human body is composed of 6 elements, i.e. oxygen, carbon, hydrogen, nitrogen, calcium and phosphorus. Human body is composed of about 60% water, 15% proteins, 15% lipids, 2% carbohydrates and 8% minerals.
In living organisms, biomolecules are ordered into a hierarchy of increasing molecular complexity.
These biomolecules are covalently linked to each other to form macromolecules of the cell, e.g. glucose to glycogen, amino acids to proteins, etc. Major complex biomolecules are Proteins, Polysaccharides, Lipids and Nucleic acids. The macromolecules associate with each other to form supramolecular systems, e.g. ribosomes, lipoproteins. Finally at the highest level of organisation in the hierarchy of cell structure, various supramolecular complexes are further assembled into cell organelle. In prokaryotes (bacteria; Greek word "pro" = before; karyon = nucleus), these macro-molecules are seen in a homogeneous matrix; but in eukaryotic cells (higher organisms; Greek word "eu" = true), the cytoplasm contains various subcellular orga-nelles. Comparison of prokaryotes and eukaryotes are shown in Table 1.2.
When the cell membrane is disrupted, the organised particles inside the cell are homogenised. This is usually carried out in 0.25 M sucrose at pH 7.4. They could then be separated by applying differential centrifugal forces. For example, the homogenate is centrifuged at 600–750 x g for 10 minutes, when nucleus and plasma membrane remnants are pelleted. On applying further centrifugal force, mitochondria are pelleted. On applying more force, lysosomes, Golgi complexes and microsomes are precipitated.
- It is the most prominent organelle of the cell. All cells in the body contain nucleus, except mature RBCs in circulation. In some cells, nucleus occupies most of the available space, e.g. small lymphocytes and spermatozoa (Fig. 1.1).
- Nucleus is surrounded by two membranes: the inner one is called perinuclear membrane with numerous pores. The outer membrane is continuous with membrane of endoplasmic reticulum.
- Nucleus contains the DNA, the chemical basis of genes which governs all the functions of the cell. The very long DNA molecules are complexed with proteins to form chromatin and chromosomes. DNA replication and RNA synthesis (transcription) are taking place inside the nucleus.
- In some cells, a portion of the nucleus may be seen as lighter shaded area; this is called nucleolus. This is the area for RNA processing and ribosome synthesis. The nucleolus is very prominent in cells actively synthesising proteins.
2. Endoplasmic Reticulum (ER)
- It is a network of interconnecting membranes enclosing channels or cisternae, that are continuous from perinuclear envelope to outer plasma membrane. Under electron microscope, the reticular arrangements will have railway track appearance (Fig. 1.1).
- This will be very prominent in cells actively synthesising proteins, e.g. immunoglobulin secreting plasma cells.
- The proteins, glycoproteins and lipoproteins are synthesised in the ER.
Table 1.2 Comparison between prokaryotic cells and eukaryotic cellsProkaryotic cellEukaryotic cellSizeSmallLarge; 1000 to 10,000 timesCell wallRigidMembrane of lipid bilayerNucleusNot definedWell definedOrganalles including mitochondria and lysosomesNilSeveralMoreover, detoxification of various drugs is done by the enzyme, cytochrome P-450 mono-oxygenase, which is present in ER.
3. Golgi Apparatus
- It may be considered as the converging area of ER. While moving through ER, carbohydrate groups are successively added to the nascent proteins (Fig. 1.1).
- These glycoproteins finally reach the Golgi area. The carbohydrate chains are further added in the Golgi prior to secretion.
- Main function of Golgi apparatus is protein sorting, packaging and secretion.
- Solid wastes of a township are usually decomposed in incinerators. Inside a cell, such a process is taking place within the lysosomes. They are bags of enzymes (Fig. 1.1).
- Lysosomes contain enzymes that hydrolyse polysaccharides, lipids, proteins, and nucleic acids.
- Endocytic vesicles and phagosomes are fused with lysosome (primary) to form the secondary lysosome or digestive vacuole. Foreign particles are progressively digested inside these vacuoles.
- They are spherical, oval or rod-like bodies, about 0.5–1 μm in diameter and up to 7 μm in length (Fig. 1.1). Erythrocytes do not contain mitochondria. The tail of spermatozoa is fully packed with mitochondria.
- Mitochondria have two membranes. The inner membrane convolutes into folds or cristae. The mitochondrial membrane contains the enzymes of electron transport chain. The fluid matrix contains the enzymes of citric acid cycle.
- Mitochondria also contain specific DNA which encodes information for synthesis for certain mitochondrial proteins. The division of mitochondria is under the command of mitochondrial DNA.
- Antibiotics inhibiting bacterial protein synthesis do not affect cellular processes, but do inhibit mitochondrial protein biosynthesis. A summary of functions of organelles is given in Tables 1.3 and 1.4.
Table 1.3 Metabolic functions of subcellular organellesNucleusDNA replication, transcriptionEndoplasmic reticulumBiosynthesis of proteins, glycoproteins, lipoproteins, drug metabolism.Golgi bodyMaturation of synthesised proteins lipids and nucleotidesLysosomeDegradation of proteins, carbohydrates, lipids and nucleotidesMitochondriaElectron transport chain, ATP generation, TCA cycle, beta oxidation of fatty acids, ketone body productionCytosolProtein synthesis, glycolysis, glycogen metabolism, transaminations, fatty acid synthesis.
6. Plasma Membrane
- The plasma membrane separates the cell from the external environment. The membranes also separate different parts of the cell from one another, so that cellular activities are compartmentalised. It has highly selective permeability properties so that the entry and exit of compounds are regulated. The membrane is very active metabolically.
- Membranes are mainly made up of lipids, proteins and small amount of carbohydrates. The contents of these compounds vary according to the nature of the membrane. The carbohydrates are present as glycoproteins and glycolipids. Phospholipids are the most common lipids present and they are amphipathic in nature. Cell membranes contain cholesterol also.
- Fluid Mosaic Model: Membranes are made up of lipid bilayer. The phospholipids are arranged in bilayers with the polar head groups oriented towards the extracellular side and the cytoplasmic side with a hydrophobic core (Fig. 1.2).
Table 1.4 Comparison of cell with a factoryPlasmamembrane:Fence with gates; gates openwhen message is receivedNucleus:Manager's officeEndoreticulum:Conveyer belt of production unitsGolgi apparatus:Packing unitsLysosomes:IncineratorsVacuoles:Lorries carrying finished productsMitochondria:Power generating unitsEach leaflet is 25 Å thick. The total thickness is about 50 to 80 Å.
- The lipid bilayer shows free lateral movement of its components, hence the membrane is said to be fluid in nature. However, the components do not freely move from inner to outer layer, or outer to inner layer (flip-flop movement is restricted). Fluidity enables the membrane to perform endocytosis and exocytosis.
- When cholesterol concentration increases, the membrane becomes less fluid on the outer surface, but more fluid in the hydrophobic core. Unsaturated cis fatty acids increase the fluidity.
- Membrane Proteins: The peripheral proteins exist on the surfaces of the bilayer. They are attached by ionic and polar bonds to polar heads of the lipids.
- The integral membrane proteins are deeply embedded in the bilayer.
- Some of the integral membrane proteins span the whole bilayer and they are called transmembrane proteins. They can serve as receptors (for hormones, growth factors, neurotransmitters), tissue specific antigens, ion channels, membrane based enzymes, etc.
Human body is supported by the skeletal system; similarly the structure of a cell is maintained by the cytoskeleton present underneath the plasma membrane. The cytoskeleton is made up of a network of microtubules and microfilaments, which contain the proteins spectrin and ankyrin. Tubules consist of polymers of tubulin
The permeability of substances across cell membranes is dependent on their solubility in lipids and not on their molecular size. Water soluble compounds are generally impermeable and require carrier mediated transport.
Transport mechanisms are classified into
- Passive transporta-i) Simple diffusiona-ii) Facilitated diffusion
- Active transport
- Ion channels allow passage of molecules in accordance with the concentration gradient
- Pumps can drive molecules against the gradient using energy.
1. Simple Diffusion
Solutes and gases enter into the cells passively. They are driven by the concentration gradient. The rate of entry is proportional to the solubility of that solute in the hydrophobic core of the membrane. Simple diffusion occurs from higher to lower concentration. This does not require any energy. But, it is a very slow process.
2. Facilitated Diffusion
This is a carrier mediated process (Fig. 1.3-C). Important features of facilitated diffusion are:
- The carrier mechanism could be saturated.
- Structurally similar solutes can competitively inhibit the entry of the solutes.
- This mechanism does not require energy but the rate of transport is more rapid than diffusion process.
- It is dependent on concentration gradient.
- Hormones regulate the number of carrier molecules. For example, glucose transport across membrane is by facilitated diffusion involving a family of glucose transporters. There are 7 different carrier mechanisms for carbohydrates and 5 different carrier systems for amino acids.
3. Ion Channels
- Cation conductive channels generally remain closed but in response to stimulus, they open allowing rapid flux of ions down the gradient. This may be compared to opening of the gate of a cinema house, when people rush to enter in. Hence this regulation is named as "gated" (Fig. 1.3-B).
- Based on the nature of stimuli that trigger the opening of the gate, they are classified into "voltage gated" or "ligand gated" ion channels. Voltage gated channels are opened by membrane depolarisation. Ligand gated channels are opened by binding of effectors.
3-a) Ligand gated channels
Acetyl choline receptor is the extensively studied example for ligand gated ion channel. It is present in post-synaptic membrane. Acetyl choline released from the presynaptic region binds with the receptors on the post-synaptic region, which triggers opening of the channel and influx of Na+. This generates an action potential in the post-synaptic nerve. The channel opens only for a millisecond, because the acetyl choline is rapidly degraded by acetyl cholinesterase.
It is a protein present in enamel of teeth has hydrophobic residues on the outside (Chapter 6). The 27 amino acid portion of amelogenin functions as a calcium channel. Phosphorylation of a serine residue of the protein opens the calcium channel, through which calcium ions zoom through and are funnelled to the mineralisation front. The calcium ions are used for the formation of calcium hydroxy apatite crystals.
3-c) Voltage Gated Channels
The channel is usually closed in the ground state. The membrane potential change (voltage difference) switches the ion channel to open, lasting less than 25 milliseconds. Voltage gated sodium channels and voltage gated potassium channels are the common examples. These are seen in nerve cells and are involved in the conduction of nerve impulses.
4. Active Transport
- The salient features of active transport are:
- This form of transport requires energy.About 40% of the total energy expenditure in a cell is used for the active transport system (Fig. 1.3-A).
- The active transport is unidirectional.
- It requires specialised integral proteins called transporters.
- The transport system is saturated at higher concentrations of solutes.
- The transporters are susceptible to inhibition by specific organic or inorganic compounds.
- Cell has low intracellular sodium; but concentration of potassium inside the cell is very high. This is maintained by the sodium–potassium activated ATPase, generally called as sodium pump. The ATPase is an integral protein of the membrane.
- The hydrolysis of one molecule of ATP can result in expulsion of 3 Na+ ions and influx of 2 K+ ions. The ion transport and ATP hydrolysis are tightly coupled.
- Clinical Applications: Cardiotonic drug digoxin inhibits the sodium–potassium pump. This leads to an increase in Na+ level inside the cell and extrusion of Ca+ from the myocardial cell. This would enhance the contractility of the cardiac muscle and so improve the function of the heart.
- Calcium Pump: The ATP dependent calcium pump also functions to regulate muscle contraction. A specialised membrane system called sarcoplasmic reticulum is found in skeletal muscles which regulates the Ca++ concentration around muscle fibers. In resting muscle the concentration of Ca++ around muscle fibres is low.
Table 1.5 Summary: transport mechanismsCarrierEnergy requiredExamplesSimple diffusionnilnilwaterFacilitated diffusionyesnilglucose to RBCsPrimary activeyesdirectsodium pumpSecondary activeyesindirectglucose to intestineIon channelsyesnosodium channelBut stimulation by a nerve impulse results in a sudden release of large amounts of Ca++. This would trigger muscle contraction. The function of calcium pump is to remove cytosolic calcium and maintain low cytosolic concentration, so that muscle can receive the next signal. For each ATP hydrolysed, 2Ca++ ions are transported.
5. Uniport, Symport and Antiport
Transport systems are classified as uniport, symport and antiport systems
- Uniport system (Fig. 1.3-D) carries single solute across the membrane, e.g., glucose transporter in most of the cells. Calcium pump is another example.
- If the transfer of one molecule depends on simultaneous or sequential transfer of another molecule, it is called co-transport system. The active transport may be coupled with energy indirectly. Here, movement of the substance against a concentration gradient is coupled with movement of a second substance down the concentration gradient; the second molecule being already concentrated within the cell by an energy requiring process.
- The cotransport system may either be a symport or an antiport. In symport (Fig. 1.3-E), the transporter carries two solutes in the same direction across the membrane, e.g., sodium dependent glucose transport. Phlorhizin, an inhibitor of sodium-dependent cotransport of glucose, especially in the proximal convoluted tubules of kidney, produces renal damage and results in renal diabetes. Amino acid transport is another example for symport.
A QUICK LOOK (CHAPTER 1)
- Cell is the basic unit of all living organisms.
- In a cell, biomolecules are maintained in a state of ‘dynamic’ or ‘steady state’ equilibrium.
- Cell organelles can be separated by density gradient ultracentrifugation. The process is known as Cell Fractionation.
- All cells in the body contain nucleus except mature erythrocytes.
- Endoplasmic reticulum is involved in protein synthesis and also detoxification of various drugs.
- Golgi apparatus is primarily involved in glycosylation, protein sorting, packaging and secretion.
- Lysosomes are packets containing many hydrolysing enzymes.
- Mitochondria are the ‘power house’ of the cell. They have their own DNA. They can synthesise their own proteins.
- Antibiotics inhibiting bacterial protein biosynthesis can inhibit mitochondrial protein biosynthesis also.
- Membranes are mainly composed of lipids (phospholipids), proteins and a small percentage of carbohydrates.
- Phospholipids, which are amphipathic in nature, are arranged as bilayers.
- Cholesterol content and nature of the fatty acid of the membrane, influence the fluidity.
- Membrane proteins can be Integral, Peripheral or Transmembrane.
- Transmembrane proteins serve as receptors, tissue specific antigens, ion-channels etc.
- Transport of molecules across the plasma membrane could be energy dependent (Active) or energy independent (Passive).
- Ion-channels function for the transport of the ions such as Ca2+, K+, Cl-, Na+ etc.
- Na+-K+ ATPase (Sodium Pump) is an example of Active transport. Cardiotonic drugs like Digoxin and Ouabain competitively inhibit K+ ion binding. The property is used to enhance contractility of the cardiac muscle.
- Transport systems may be Uniport, Antiport or Symport.