De Jesus, Ardette S. Biochemistry Section B1 CLINICAL CASE #1 BOTULISM 1. Discuss the functional unit of muscle and its myofibrils.

Answer: Each skeletal muscle fiber contains bundles of filaments, called myofibrils, running along the axis of the cell. It has a diameter of 1-2 um and consist of an end to end repetitive arrangement of sarcomeres. A myofibril can be subdivided longitudinally into SARCOMERE, the contractile unit in myofiber. Sarcomere is composed of myofilaments (contractile proteins) arranged in thick and thin filaments. The sarcomere is demarcated with two dark lines called Z lines and represents a repeating contractile unit in skeletal muscle. Light band (I band) contains thin filaments composed primarily of the protein actin. Dark band (A band) contains thick filaments composed primarily of the protein myosin. A light area present the in the center of the sarcomere is called the H band. A dark line called the M line is evident in the center of the sarcomere and includes proteins that appear to be critical for organization and alignment of the thick filaments in the sarcomere

2. What are the major proteins of muscle and their functions. Answer: a. ACTIN- consists of long filamentous polymers containing two strands of globular (G-actin) monomers, 5-6 nm in diameter, twisted around each other in a double helical formation. It is asymmetric and polymerize to produce a filament with polarity. It contains binding site for myosin. b. TROPONIN- consists of three subunits (Tt, Ti,Tc); it is present on each tropomyosin dimer and influences the position of the tropomyosin molecule on the actin filament and hence the ability of tropomyosin to inhibit binding of myosin to the actin filament. c. TROPOMYOSIN- dimer of the protein; extend over the entire actin filament and cover myosin binding sites on the actin molecule. d. MYOSIN- is large protein that consists of six different polypeptides with one pair of large heavy chains and two pairs of light chains. The head region extends away from the thick filament toward

the actin thin filament and is the portion of the molecule that can bind to actin. It is also able to hydrolyze ATP, and ATPase activity is located in the globular head as well. 3. What are the major biochemical events occurring during a muscle contraction and relaxation? Answer: A nerve impulse triggers the release of Ach from the synaptic knob into the synaptic cleft. Ach binds to Ach receptors in the motor end plate of the neuromuscular junction, initiating a muscle impulse in the sarcolemma of the muscle fiber. As the muscle impulse spreads quickly from the sarcolemma along T-tubules, calcium ions are released from terminal cisternae into the sarcoplasm. Calcium ions bind to troponin. Troponin changes shape, moving tropomyosin on the actin to expose active sites on actin molecules of thin filaments. Myosin heads of thick filaments attach to exposed active sites to form crossbridges. Myosin heads pivot, moving thin filaments toward the sarcomere center. ATP binds myosin heads and is broken down into ADP and P. Myosin heads detach from thin filaments and return to their pre-pivot position.

4. Discuss the synthesis, upgrade and degradation of acetylcholine in neuromuscular junction. Answer: ----In the axon terminal are many mitochondria that supply adenosine triphosphate (ATP), the energy source that is used for synthesis of an excitatory transmitter acetylcholine. The acetylcholine in turn excites the muscle fiber membrane. Acetylcholine is synthesized in the cytoplasm of the terminal, but it is absorbed rapidly into many small synaptic vesicles about 300,00 of which are normally in the terminals of a single end plate. In the synaptic space are large quantities of the enzyme acetylcholinesterase, which destroys acetylcholine a few milliseconds after it has been released from the synaptic vesicles. (Guyton, page 85). ---- Synthesis of acetylcholine is facilitated by the enzyme, choline acetyltransferase (CAT). This enzyme combines choline with acetate derived from acetyl coenzyme A (CoA). ACh in cholinergic nerve fibers is taken up into synaptic vesicles by an uptake process. Release of acetylcholine, like synaptic release at other junctions, is based on quantal release of vesicles containing preformed neurotransmitter molecules. Vesicular release depends on depolarization of the nerve terminal and the influx of calcium ion. Acetylcholine (ACh) is terminated by hydrolysis, which is greatly accelerated by one or more of the cholinesterase enzymes. Acetylcholinesterase (AChE) is present in high concentration in cholinergic synapses. (http://courses.washington.edu/chat543/cvans/sfp/acetylch.html)

5. Describe the structure of Botulinum toxin, and how it is activated. * The light chain (~50 kD - amino acids 1-448) acts as a zinc (Zn2+) endopeptidase similar to tetanus toxin with proteolytic activity located at the N-terminal end (see image below). The heavy chain (~100 kD - amino acids 449-1280) provides cholinergic specificity and is responsible for binding the toxin to presynaptic receptors; it also promotes light-chain translocation across the endosomal membrane. *A. BINDING TO RECEPTORS ON UNMYELINATED PRESYNAPTIC MEMBRANE (DOUBLE RECEPTOR) B. UPTAKE OF TOXIN INTO NERVE TERMINALS BY ENDOCYTOSIS C. TRANSLOCATION TO CYTOSOL D. INHIBITION OF TRANSMITTER EXOCYTOSIS FROM PRESYNAPTIC TERMINAL *BLOCK NT RELEASE AT PERIPHERAL CHOLINERGIC NERVE TERMINALS

SYMPATHETIC AND PARASYMPATHETIC * Botulinum toxin acts by binding presynaptically to high-affinity recognition sites on the cholinergic nerve terminals and decreasing the release of acetylcholine, causing a neuromuscular blocking effect.

6. How does Botulinum toxin cause FOOD poisoning? Discuss its main role in the pathophysiology of the symptoms shown in the case. Answer: 1. PASSAGE OF TOXIN FROM GI TRACT TO VASCULATURE 2. TOXIN PASSES OUT OF VASCULATURE TO PRESYNAPTIC REGIONS A. BINDING TO RECEPTORS ON UNMYELINATED PRESYNAPTIC MEMBRANE (DOUBLE RECEPTOR) B. UPTAKE OF TOXIN INTO NERVE TERMINALS BY ENDOCYTOSIS C. TRANSLOCATION TO CYTOSOL D. INHIBITION OF TRANSMITTER EXOCYTOSIS FROM PRESYNAPTIC TERMINAL * Clinical botulism results from the entry of botulinum toxin into the systemic circulation and subsequent inhibition of acetylcholine release from the presynaptic nerve terminal. The toxin enters the circulation through the mucosa (food-borne and inhalational) or via a break in the skin (wound and iatrogenic). Once absorbed into the bloodstream, the toxin is carried to the synapses of peripheral and cranial nerves. The heavy chain mediates binding of the toxin to presynaptic receptors, allowing for receptor-mediated endocytosis. Acetylcholine release at the neuromuscular junction is mediated by a synaptic-fusion complex. This complex consists of 3 soluble fusion attachment protein receptors (SNARE proteins). The toxin light chain inhibits vesicle release by cleaving peptide bonds of these SNARE proteins. As a result, stimulation of the presynaptic cell fails to produce transmitter release, resulting in motor paralysis or autonomic dysfunction when parasympathetic nerve terminals or autonomic ganglia are involved.