1.1: Structure and Functions of the Cell and Cellular Organelles

(Estimated Time: 1 Hour)

Objective: To understand the eukaryotic cell as a highly organized system of compartments, each with a specific structure that dictates its unique biochemical functions. This knowledge forms the basis for understanding metabolic pathways and the molecular basis of disease.

I. The Eukaryotic Cell: A System of Compartments

A eukaryotic cell is defined by the presence of a true nucleus and other membrane-bound organelles. This compartmentalization is a critical principle in biochemistry because it:

• Increases Efficiency: Concentrates enzymes and substrates for specific metabolic pathways.

• Separates Incompatible Reactions: Allows simultaneous anabolic (synthesis) and catabolic (breakdown) processes without interference (e.g., fatty acid synthesis in the cytosol vs. fatty acid oxidation in the mitochondria).

• Establishes Unique Environments: Creates specialized conditions, such as the low pH of lysosomes or the proton gradient across the inner mitochondrial membrane.

II. The Plasma Membrane: The Dynamic Interface

The plasma membrane is not just a passive barrier but a dynamic, semi-permeable structure crucial for transport, communication, and cell identity.

• A. Structure: The Fluid Mosaic Model

  1. Lipid Bilayer (The "Fluid" part):

     • Phospholipids: The fundamental building blocks. They are amphipathic, having a hydrophilic (water-loving) phosphate head and two hydrophobic (water-fearing) fatty acid tails. They spontaneously form a bilayer in aqueous environments, with heads facing outwards and tails inwards.

     • Cholesterol: Interspersed between phospholipids. Its crucial biochemical role is to modulate membrane fluidity. At high temperatures, it restrains phospholipid movement, making the membrane less fluid. At low temperatures, it prevents packing, making the membrane more fluid (prevents crystallization). This is vital for maintaining the function of membrane proteins.

     • Glycolipids: Lipids with attached carbohydrate chains, found exclusively on the outer leaflet. They are important in cell-cell recognition and act as receptors (e.g., receptors for cholera toxin).

  2. Membrane Proteins (The "Mosaic" part):

     • Integral (Intrinsic) Proteins: Tightly embedded within the bilayer, often spanning it entirely (transmembrane proteins). They have hydrophobic regions that interact with the fatty acid tails.

       • Examples & Functions:

         • Channels: Form pores for rapid passage of water (Aquaporins) or ions.

         • Transporters/Carriers: Bind a substance and undergo a conformational change to move it across the membrane (e.g., Glucose Transporters - GLUTs).

         • Pumps: Use energy (ATP) to move substances against their concentration gradient (e.g., Na⁺-K⁺ ATPase).

         • Receptors: Bind to signaling molecules (ligands) like hormones or neurotransmitters, initiating an intracellular response (e.g., G-Protein Coupled Receptors - GPCRs).

• 2. • Peripheral (Extrinsic) Proteins: Loosely bound to the surface of the membrane (either inner or outer), often attached to integral proteins.

       • Examples & Functions:

         • Spectrin and Ankyrin: Found on the inner surface of the red blood cell membrane, they form a cytoskeletal mesh that maintains the cell's biconcave shape. Defects lead to inherited anemias like hereditary spherocytosis.

         • Cytochrome c: A peripheral protein on the outer surface of the inner mitochondrial membrane, crucial for the electron transport chain.

• B. Key Biochemical Functions of the Plasma Membrane

  1. Selective Permeability & Transport:

     • Passive Transport (No energy required):

       • Simple Diffusion: Small, nonpolar molecules (O₂, CO₂, steroid hormones) move down their concentration gradient.

       • Facilitated Diffusion: Requires a carrier or channel protein (e.g., glucose entry into most cells via GLUT transporters). Rate is saturable.

     • Active Transport (Energy required):

       • Primary Active Transport: Directly uses ATP. The Na⁺-K⁺ ATPase pump is the classic example. It pumps 3 Na⁺ out and 2 K⁺ in per ATP hydrolyzed, establishing critical electrochemical gradients for nerve impulses and secondary active transport.

       • Secondary Active Transport: Uses the energy stored in an ion gradient (like the Na⁺ gradient) to transport another molecule. Example: Sodium-Glucose Linked Transporter (SGLT1) in the intestine and kidney uses the inward flow of Na⁺ to co-transport glucose against its concentration gradient. This is the principle behind Oral Rehydration Solution (ORS), which contains both glucose and salt to maximize water and electrolyte absorption.

  2. Signal Transduction: Binding of a hormone (e.g., glucagon, insulin) to its specific receptor initiates a cascade of intracellular events, altering cell metabolism.

  3. Cell Recognition: The glycocalyx (the carbohydrate coat on the outer surface) acts as a cellular "ID card." The ABO blood group antigens are carbohydrate structures on glycolipids and glycoproteins of red blood cells.

III. Organelles of Biosynthesis and Processing

• A. The Nucleus: The Information Storehouse

  • Structure: Surrounded by a double membrane (nuclear envelope) perforated by nuclear pores which regulate the passage of molecules (e.g., mRNA out, nuclear proteins in). Contains chromatin (DNA complexed with histone proteins) and a nucleolus.

  • Biochemical Functions:

    1. DNA Replication: Duplication of the entire genome before cell division.

    2. Transcription: Synthesis of RNA (mRNA, tRNA, rRNA) from a DNA template.

    3. Ribosome Synthesis: The nucleolus is the site of ribosomal RNA (rRNA) synthesis and assembly of ribosomal subunits.

• B. Ribosomes: The Protein Synthesizers

  • Structure: Non-membranous particles composed of rRNA and proteins. Eukaryotic ribosomes are 80S (composed of 60S and 40S subunits).

  • Biochemical Function: Site of translation (protein synthesis).

  • Two Populations:

    1. Free Ribosomes (in cytosol): Synthesize proteins destined for the cytosol, nucleus, mitochondria, and peroxisomes.

    2. Bound Ribosomes (on RER): Synthesize proteins destined for secretion, insertion into membranes, or delivery to lysosomes.

• C. Endoplasmic Reticulum (ER): The Biosynthetic Factory

  • Rough ER (RER): Studded with ribosomes.

    1. Function: Synthesis and modification of proteins from bound ribosomes. Key modifications include:

       • N-linked Glycosylation: Addition of a pre-formed oligosaccharide chain to the asparagine (N) residue of a growing polypeptide.

       • Protein Folding: Assisted by chaperone proteins like BiP (Binding immunoglobulin Protein).

• • Smooth ER (SER): Tubular network lacking ribosomes.

    1. Function:

       • Lipid Synthesis: Site of synthesis for steroids (e.g., cholesterol, cortisol), triglycerides, and phospholipids. Abundant in cells of the adrenal cortex and liver.

       • Detoxification: Contains the Cytochrome P450 (CYP) enzyme system in the liver. These enzymes perform Phase I detoxification reactions, typically adding a hydroxyl (-OH) group to make nonpolar drugs and toxins more water-soluble for excretion.

       • Calcium Storage: The sarcoplasmic reticulum in muscle cells is a specialized SER that stores and releases Ca²⁺ to trigger muscle contraction.

• D. Golgi Apparatus: The Finishing and Shipping Center

  • Structure: A stack of flattened, membrane-bound sacs called cisternae. It has polarity: a cis-face (receives vesicles from the RER) and a trans-face (ships vesicles out).

  • Biochemical Functions:

    1. Further Protein Modification:

       • O-linked Glycosylation: Addition of sugars to serine (S) or threonine (T) residues.

       • Modification of N-linked oligosaccharides added in the RER.

    2. Sorting and Packaging: Sorts proteins into different transport vesicles destined for the plasma membrane (secretion), lysosomes, or other organelles. The mannose-6-phosphate (M6P) tag is a specific biochemical signal added in the Golgi to direct enzymes to the lysosome.

IV. Organelles of Degradation and Detoxification

• A. Lysosomes: The Recycling Center

  • Structure: Single-membrane vesicles containing over 50 different acid hydrolases (proteases, lipases, nucleases, etc.).

  • Biochemical Environment: Maintain an internal acidic pH of ~5.0. This is achieved by a V-type proton pump (H⁺-ATPase) in the lysosomal membrane, which actively pumps H⁺ into the lumen. This acidic pH is optimal for the hydrolytic enzymes and protects the cell from accidental leakage (these enzymes are largely inactive at the neutral pH of the cytosol).

  • Function: Intracellular digestion of material brought in by endocytosis, phagocytosis, and autophagy (digestion of old organelles).

• B. Peroxisomes: The Specialized Oxidation Center

  • Structure: Single-membrane vesicles containing oxidative enzymes like catalase and urate oxidase.

  • Biochemical Functions:

    1. Beta-oxidation of Very-Long-Chain Fatty Acids (VLCFAs): Fatty acids with >22 carbons are shortened in peroxisomes before being fully oxidized in mitochondria.

    2. Detoxification:

       • Breakdown of hydrogen peroxide (H₂O₂), a toxic byproduct of oxidative reactions, via the enzyme Catalase: 2 H₂O₂ → 2 H₂O + O₂.

       • Oxidation of ethanol to acetaldehyde.

    3. Synthesis of Plasmalogens: A class of ether-phospholipids essential for the structure of myelin in nerve cells.

V. The Mitochondrion: The Powerhouse of the Cell

• Structure: Unique double-membrane structure.

  1. Outer Membrane: Smooth and permeable to small molecules and ions.

  2. Inner Membrane: Highly folded into cristae to dramatically increase surface area. It is impermeable to most ions, requiring specific transporters. It is the site of the Electron Transport Chain (ETC) and ATP synthase.

  3. Matrix: The innermost compartment, containing enzymes for the TCA cycle, beta-oxidation, parts of the urea cycle, and its own circular DNA and ribosomes.

• Key Biochemical Functions:

  1. Energy Transduction: The central hub for ATP production via cellular respiration. This involves the coordinated action of the TCA cycle (matrix), ETC (inner membrane), and oxidative phosphorylation (inner membrane).

  2. Fatty Acid Oxidation (Beta-oxidation): Occurs in the matrix.

  3. Initiation of Apoptosis (Programmed Cell Death): Under certain stress signals, the mitochondria release cytochrome c from the intermembrane space into the cytosol, triggering a cascade of caspase enzymes that leads to cell death. This is a critical process in development and disease.

1.2: Cell Fractionation, Marker Enzymes, and Diseases of Organelles

(Estimated Time: 1 Hour)

Objective: To understand the experimental techniques used to isolate and identify subcellular organelles and to correlate defects in organelle function with specific human diseases, providing a molecular basis for clinical pathology.

I. Techniques for Studying Organelles: The "How We Know"

Before we can understand diseases of organelles, we must first understand how biochemists isolate and study them.

• A. Cell Fractionation: Separating the Components

  • Purpose: To isolate a specific type of organelle in a pure, functional form, allowing for the study of its unique biochemical activities (e.g., studying ATP synthesis in isolated mitochondria).

  • Principle: Separation based on physical properties: size, shape, and density. Larger, denser organelles sediment (form a pellet) at lower centrifugal forces than smaller, less dense ones.

  • Method: Differential Centrifugation
    This is a sequential process performed in a cold, isotonic, and buffered solution to preserve organelle integrity and function.

    1. Step 1: Homogenization: Tissues (e.g., liver) are gently disrupted in a blender or homogenizer. This breaks the plasma membranes, releasing the organelles into the buffer, creating a mixture called the homogenate.

    2. Step 2: Low-Speed Centrifugation (e.g., 600 x g for 10 min): The homogenate is spun. The largest and densest components pellet at the bottom.

       • Pellet 1 (Nuclear Fraction): Contains whole cells, nuclei, and the cytoskeleton.

       • Supernatant 1: Contains all other smaller organelles.

    3. Step 3: Medium-Speed Centrifugation (e.g., 15,000 x g for 20 min): The supernatant from the previous step is transferred to a new tube and spun at a higher speed.

       • Pellet 2 (Mitochondrial Fraction): Contains mitochondria, lysosomes, and peroxisomes.

       • Supernatant 2: Contains even smaller components.

    4. Step 4: High-Speed Centrifugation (e.g., 100,000 x g for 60 min): The second supernatant is spun at a very high speed.

       • Pellet 3 (Microsomal Fraction): This pellet contains microsomes, which are small, sealed vesicles formed from fragments of the endoplasmic reticulum and Golgi apparatus.

       • Supernatant 3: Contains the soluble components of the cell.

    5. Step 5: Ultracentrifugation (e.g., >100,000 x g for several hours): The final supernatant is spun at extremely high speeds.

       • Pellet 4: Contains ribosomes and other large macromolecules.

       • Final Supernatant: This is the cytosol, the soluble part of the cytoplasm, containing enzymes of glycolysis, soluble proteins, etc.

• B. Marker Enzymes: The Biochemical "Address Labels"

  • Concept: A marker enzyme is an enzyme that is uniquely or predominantly found within a single type of organelle.

  • Clinical and Research Utility:

    1. Identification: Assaying for the activity of a marker enzyme in a fraction confirms the presence of that organelle.

    2. Purity Assessment: The specific activity (activity per mg of protein) of the marker enzyme will be highest in the pure organelle fraction. Contamination is indicated by the presence of marker enzymes from other organelles.

  • Essential Marker Enzymes to Memorize:

Organelle Marker Enzyme Biochemical Significance of the Enzyme

Plasma Membrane Na⁺-K⁺ ATPase Primary active transport pump

Mitochondria Succinate Dehydrogenase Enzyme of both TCA Cycle & ETC (Complex II)

Lysosome Acid Phosphatase A key acid hydrolase

Endoplasmic Reticulum Glucose-6-Phosphatase Final step of gluconeogenesis (in liver/kidney)

Golgi Apparatus Galactosyl Transferase Adds galactose during glycosylation

Peroxisome Catalase Detoxifies hydrogen peroxide

Cytosol Lactate Dehydrogenase (LDH) Key enzyme in anaerobic glycolysis

Nucleus DNA Polymerase Essential for DNA replication

II. Organelle Pathologies: The Clinical Correlation

When these highly specialized compartments fail, devastating diseases result.

• A. Lysosomal Storage Diseases (LSDs)

  • General Pathophysiology: A group of ~50 inherited metabolic disorders caused by a deficiency of a single specific lysosomal acid hydrolase. This leads to the progressive accumulation of the enzyme's substrate within the lysosome. The engorged lysosomes disrupt normal cell function, leading to cell death and organ damage.

  • High-Yield Clinical Examples:

    1. Tay-Sachs Disease:

       • Biochemical Defect: Deficiency of Hexosaminidase A.

       • Accumulated Substrate: GM₂ ganglioside (a lipid found in nerve cell membranes).

       • Clinical Features: Rapid and progressive neurodegeneration, developmental regression after 3-6 months of age, profound muscle weakness (hypotonia), and a characteristic cherry-red spot on the macula of the retina.

    2. Gaucher's Disease:

       • Biochemical Defect: Deficiency of Glucocerebrosidase (β-glucosidase).

       • Accumulated Substrate: Glucocerebroside.

       • Clinical Features: Massive hepatosplenomegaly, bone pain and fractures, anemia, and thrombocytopenia. The classic histological finding is the Gaucher cell – a macrophage engorged with lipid, having a "crumpled tissue paper" appearance.

    3. I-Cell Disease (Mucolipidosis II) - A Disease of Protein Targeting:

       • Biochemical Defect: This is a unique LSD. The lysosomal enzymes themselves are normal, but they fail to be targeted to the lysosome. The defect is in the enzyme N-acetylglucosamine-1-phosphotransferase in the Golgi apparatus. This enzyme is responsible for adding the mannose-6-phosphate (M6P) tag to the enzymes.

       • Pathophysiology: Without the M6P "address label," the newly synthesized acid hydrolases are mistakenly secreted out of the cell instead of being delivered to lysosomes.

       • Cellular and Clinical Features: Lysosomes are empty of enzymes but full of undigested material (Inclusion cells or "I-cells"). Clinically, it presents with coarse facial features, skeletal abnormalities, and severe psychomotor retardation. A key diagnostic finding is high levels of lysosomal enzymes in the patient's plasma.

• B. Peroxisomal Disorders

  • General Pathophysiology: Result from either a failure to assemble functional peroxisomes (biogenesis disorders) or a deficiency in a single peroxisomal enzyme.

  • High-Yield Clinical Examples:

    1. Zellweger Syndrome:

       • Biochemical Defect: A defect in peroxins (PEX genes), proteins required for peroxisome assembly. This results in "empty" or absent peroxisomes.

       • Pathophysiology: Multiple peroxisomal functions fail, most notably the oxidation of Very-Long-Chain Fatty Acids (VLCFAs).

       • Clinical Features: Severe neurological abnormalities (hypotonia, seizures), distinctive craniofacial features, and liver dysfunction. VLCFAs accumulate in blood and tissues.

    2. X-linked Adrenoleukodystrophy (X-ALD):

       • Biochemical Defect: A defect in a specific peroxisomal membrane transporter protein (ABCD1).

       • Pathophysiology: VLCFAs cannot be transported into the peroxisome for degradation. They accumulate and are incorporated into cell membranes, where they disrupt the structure and function of myelin in the central nervous system and the adrenal cortex.

       • Clinical Features: Adrenal insufficiency (Addison's disease) and progressive neurological demyelination leading to dementia, spasticity, and death.

• C. Mitochondrial Diseases (Mitochondriopathies)

  • General Pathophysiology: Caused by mutations in either mitochondrial DNA (mtDNA) or nuclear DNA (nDNA) that code for proteins of the electron transport chain or ATP synthase. The result is a severe deficiency in ATP production.

  • Key Genetic Concepts:

    1. Maternal Inheritance: mtDNA is inherited exclusively from the mother via the oocyte. An affected male does not pass the disease to his children.

    2. Heteroplasmy: A cell contains many mitochondria. A mixture of mutant and normal mtDNA can exist within a single cell. The clinical severity of the disease often depends on the ratio of mutant to normal mtDNA.

  • High-Yield Clinical Examples: (Tissues with high energy demand are most affected).

    1. Leber's Hereditary Optic Neuropathy (LHON):

       • Biochemical Defect: Typically a point mutation in an mtDNA gene for a subunit of ETC Complex I.

       • Clinical Features: Acute, painless, bilateral loss of central vision, primarily affecting young adult males.

    2. MERRF (Myoclonic Epilepsy with Ragged Red Fibers):

       • Biochemical Defect: Mutation in an mtDNA tRNA gene, impairing mitochondrial protein synthesis.

       • Clinical Features: Myoclonus (brief, involuntary muscle twitching), seizures, ataxia. The name derives from the characteristic histological finding on muscle biopsy: "ragged red fibers", which are muscle cells with accumulations of abnormal mitochondria that stain red with Gomori trichrome stain.

• D. Diseases of the Endoplasmic Reticulum and Protein Folding

  • General Pathophysiology: The RER has a stringent quality control system. If a protein is misfolded, it is retained in the ER and targeted for degradation. If misfolded proteins accumulate, it triggers a state of ER stress and the Unfolded Protein Response (UPR).

  • High-Yield Clinical Example:

    1. Cystic Fibrosis (CF):

       • Biochemical Defect: Most commonly caused by a three-base-pair deletion in the CFTR gene, leading to the loss of a phenylalanine residue at position 508 (ΔF508 mutation).

       • Pathophysiology: The mutant CFTR protein (a chloride ion channel) misfolds within the RER. The cell's quality control machinery recognizes it as faulty and targets it for premature degradation via the ubiquitin-proteasome system. Consequently, the channel never reaches the plasma membrane.

       • Clinical Features: The absence of the chloride channel in epithelial cells leads to defective ion transport and abnormally thick, viscous mucus in the lungs (recurrent infections), pancreas (malabsorption), and other organs.