Q1 LONG ANSWER 10M ★ High-yield

Describe the structure and functions of the cell organelles. Give their marker enzymes and the principle of subcellular fractionation.

Biochemistry · 1st Professional — Pre-Clinical

Introduction

The cell is the structural and functional unit of life. Metabolic pathways are compartmentalised among its organelles, which allows incompatible reactions to proceed simultaneously and permits independent regulation.

The Organelles and Their Functions

Organelle	Chief functions
Nucleus	DNA replication and transcription; the nucleolus makes ribosomal RNA
Mitochondrion	TCA cycle, electron transport, oxidative phosphorylation, β-oxidation, ketogenesis, part of the urea cycle, PDH complex
Rough endoplasmic reticulum	Synthesis of secretory and membrane proteins
Smooth endoplasmic reticulum	Lipid and steroid synthesis; detoxification (cytochrome P450); calcium store
Golgi apparatus	Glycosylation, sulphation, sorting and packaging of proteins
Lysosome	Intracellular digestion by about 50 acid hydrolases at pH 5
Peroxisome	Oxidation of very long chain fatty acids, α-oxidation, plasmalogen synthesis, catalase
Ribosome	Protein synthesis (80S in eukaryotes)
Cytosol	Glycolysis, HMP shunt, fatty acid synthesis, glycogen metabolism
Cytoskeleton	Shape, motility and intracellular transport — microfilaments (actin), microtubules (tubulin), intermediate filaments

Marker Enzymes

A marker enzyme is one confined to a single organelle, used to identify and assess the purity of fractions after subcellular fractionation.

Organelle	Marker enzyme
Mitochondrion	Cytochrome oxidase, succinate dehydrogenase, glutamate dehydrogenase
Lysosome	Acid phosphatase, β-glucuronidase
Endoplasmic reticulum (microsomes)	Glucose-6-phosphatase, cytochrome P450
Golgi	Galactosyl transferase
Peroxisome	Catalase, urate oxidase
Cytosol	Lactate dehydrogenase
Plasma membrane	Na+/K+-ATPase, 5′-nucleotidase, adenylate cyclase
Nucleus	DNA polymerase, RNA polymerase

Subcellular Fractionation

Tissue homogenised in isotonic sucrose → Differential centrifugation at increasing speed → 600 × g → nuclei → 10,000 × g → mitochondria, lysosomes, peroxisomes → 100,000 × g → microsomes (fragmented ER) → Supernatant → cytosol

Density gradient centrifugation separates organelles of similar size but different density
Purity is judged by marker enzyme assay — the practical reason markers matter

Mitochondria in Detail

Double membrane; the outer is permeable (porins), the inner impermeable and folded into cristae
Inner membrane — electron transport chain, ATP synthase, carrier proteins; rich in cardiolipin
Matrix — TCA cycle, β-oxidation, PDH complex, urea cycle enzymes
Own circular DNA (16.5 kb), its own ribosomes (70S) and tRNAs
Maternally inherited — sperm mitochondria are degraded after fertilisation
Regarded as descended from an engulfed bacterium — the endosymbiont theory, supported by the double membrane, circular DNA and 70S ribosomes

Lysosomes

Single membrane; interior kept at pH 5 by an H+-ATPase
Contain about 50 acid hydrolases — proteases, lipases, nucleases, glycosidases, sulphatases
Called "suicide bags"; rupture causes autolysis
Enzymes are tagged with mannose-6-phosphate in the Golgi for delivery
Functions — digestion of engulfed material, autophagy, turnover of organelles, bone remodelling by osteoclasts

Compartmentation of Metabolic Pathways

Pathway	Compartment
Glycolysis, HMP shunt, fatty acid synthesis	Cytosol
TCA cycle, β-oxidation, etc, ketogenesis	Mitochondrion
Gluconeogenesis	Both — starts in mitochondrion, ends in ER
Urea cycle	Both — first 2 steps mitochondrial, last 3 cytosolic
Haem synthesis	Both
Cholesterol and steroid synthesis	Cytosol and smooth ER
Protein synthesis	Ribosomes — free or on rough ER

Compartmentation permits opposing pathways to run at once — fatty acid synthesis in the cytosol while oxidation proceeds in the mitochondrion — and allows each to be regulated separately

Applied Aspects

Lysosomal storage diseases — deficiency of a single hydrolase causes accumulation of its substrate: Tay–Sachs, Gaucher, Niemann–Pick, Hurler, Pompe
I-cell disease — failure to add the mannose-6-phosphate tag, so enzymes are secreted instead of delivered; lysosomes are empty and plasma hydrolase levels are high
Mitochondrial diseases are maternally inherited and strike high-energy tissues — melas, MERRF, Leber hereditary optic neuropathy
Zellweger syndrome — absent peroxisomes; very long chain fatty acids accumulate
Marker enzymes in plasma indicate the site of cell damage — a rise in mitochondrial ast suggests severe hepatocyte necrosis rather than mere membrane leak

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