Enzyme Histochemistry (IH) Flashcards
Epimysium
Collagenous tissue sheath that covers an entire muscle
Perimysium
Dense layer of collagen that covers bundles of muscle fibers to form a fascicle
Endomysium
Fine collagen fibers that surround individual muscle fibers
Enzymes
Proteins that catalyze (change the rate of) chemical reactions by temporarily combining with their substrate (target)
Oxidation
Addition of oxygen
loss of hydrogen
loss of electrons
Reduction
Loss of oxygen
gain of hydrogen
gain of electrons
Hydrolase
An enzyme that typically adds (or sometimes removes) water
Includes: esterases, phosphatases, peptidases
Simultaneous Coupling
Enzyme acts on substrate and a reaction product is released
Then the reaction product is rapidly captured or coupled with a diazonium salt to yield an insoluble azo dye, or captured with a metallic ion
Esterase
Enzymes that break bonds between carboxylic acid and alcohols, phenol, and napthols.
Most are capable of hydrolyzing a-napthyl cetate
Phosphatase
hydrolytic enzymes that break the bond between an alcohol and phosphate group
found in lysosomes (acidic and alkaline versions)
ATPase is in mitochondria and bound to myosin for muscle contraction
Phosphorylase
A type of transferase that move phosphate groups
Used for synthesis and decomposition of saccharides such as glycogen
What are 3 properties of enzymes?
Act as catalysts to promote or inhibit a reaction
They are specific to the shape of a substrate based on chemical groups and orientation
They are sensitive to temperature and pH to function
What are the 6 basic enzyme reactions?
Oxidoreductases
Transferases
Hydrolases
Lyases
Isomerases
Ligases
What are 5 factors that affect enzyme demonstration?
Treatment before and during procedure (fixation/freeing/thawing)
Nonoptimal substrate (poor solubility or hydrolyzation)
Nonoptimal temperature (enzymes denatured at 56C)
Nonoptimal pH (pH7 is optimal wit some exceptions)
Inhibitors (excess diazonium salts, heat, and some metallic ions)
What is the artifact that may be seen in frozen sections?
freezing artifact, disruption of tissue? Looks like lots of little holes that disrupt morphology
How to preserve tissue for enzyme studies?
Need to be fixed before freezing to prevent diffusion artifact
What is the preferred method for freeing muscle tissue
isopentane suspended in liquid nitrogen, because it causes less ice-crystal freezing artifact
Summarize enzyme-substrate chemical reaction
Enzyme + Substrate -> temporarily combine-> yields Product + Enzyme
α-naphthyl acetate esterase reaction
a-napthyl acetate + water in presence of esterases yields -> a-napthol + acetic acid
a-napthol + hexazotized pararosaniline (basic fuchsin) yields -> azo dye for detection
non-specific esterase stain for differentiating type II atrophy and neurogenic atrophy
Naphthol AS-D choloracetate esterase reaction
a-napthyl acetate + water in presence of esterases yields -> a-napthol + acetic acid
a-napthol + hexazotized pararosaniline (basic fuchsin) yields -> azo dye for detection
This is a specific esterase stain, the only one that can be done on paraffin, and it is used to identify granulocytes for classifying leukemia’s
ATPase reaction
differentiate type I, type IIA, type IIB fibers
Gomori-type metal precipitation of the hydrolyzed orthophosphoric acid with calcium. A complex series of reactions leads to the formation of a colored metallic sulfide
(ATP + H2O) in the presence of ATPase yields -> (ADP + orthophosphate + energy)
orthophosphate combines with calcium to form calcium phosphate
calcium is exchanged with cobalt
cobalt is exchanged with sulfide to form visible insoluble cobaltous sulfide
Acid phosphatase reaction
Presence of inflammatory cells, marker for lysosomes
phosphomonoesterases hydrolyze esters of orthophosphoric acid at an acidic pH
Alcohol residue of the napthol AS-BI-phosphate reacts with hexazotized pararosaniline (basic fuchsin) to yield a red azo dye via simultaneous coupling method
NADH diaphorase reaction
Identifies abnormal mitochondria, z-bands, and sarcoplasmic reticulum
NADH (reduced) in the presence of diaphorase yields -> NAD+ (oxidized) + hydrogen
Nitro blue tetrazolium (NBT) + hydrogen yields -> formazan
SDH Succinic dehydrogenase reaction
Identify mitochondria only from NADH diaphorase activity
SDH is mitochondrial and is part of the Krebs cycle
succinate in the presence of succinic dehydrogenase yields -> fumarate + hydrogen
NBT + hydrogen yields -> formazan
