Enzymes: Lactate Dehydrogenase and Creatine Kinase Flashcards
What reaction does Lactate Dehydrogenase catalyze?
The oxidation of lactate to pyruvate or pyruvate to lactate
How many pH optimums does the Lactate Dehydrogenase reaction have?
2
What is the pH optimum for the forward LD reaction?
8.3-8.9
What is the pH optimum for the reverse LD reaction?
7.1-7.4
What is the required co-factor for the LD reaction?
NAD+
The LD reaction can occur in both directions, which direction does this reaction favor?
Reverse (pyruvate to lactate)
Is lactate dehydrogenase specific for lactate and pyruvate?
Not absolute specificity. Can also reduce a number of alpha ketoacids and oxidize several alpha hydroxyacids
What is the molecular structure of lactate dehydrogenase?
Tetramer (4 polypeptide chains) bound together by disulphide bonds
What are the 2 possible LD subunit types?
H and M subtypes
What are some LD inhibitors?
Borate, oxalate, EDTA, pyruvate, lactate, reagents with reactivity against thiol groups
How do borate and oxalate inhibit LD enzymatic activity?
They compete with lactate for the enzyme binding site
How does EDTA inhibit LD enzymatic activity?
It binds with zinc, which acts as an LD activator.
How do pyruvate and lactate inhibit LD enzymatic activity?
They inhibit due to excess
Why would serum LD be increased?
Heart disease, liver disease, muscle disease, hemolytic disorders, pulmonary disease, cancer
Name H and/or M subunits of LD-1.
H4
Name H and/or M subunits of LD-2.
H3M
Name H and/or M subunits of LD-3.
H2M2
Name H and/or M subunits of LD-4.
HM3
Name H and/or M subunits of LD-5.
M4
How many LD isoenzymes are there?
5 isoenzymes
Which LD isoenzyme(s) will be increased when heart disease is present?
LD-1 and LD-2
Which LD isoenzyme(s) will be increased when liver disease is present?
LD-4 and LD-5
Which LD isoenzyme(s) will be increased when muscle disease is present?
LD-5
Which LD isoenzyme(s) will be increased when hemolytic disorders are present?
LD-1 and LD-2
Which LD isoenzyme(s) will be increased when pulmonary disease is present?
LD-3
Which LD isoenzyme(s) will be increased when cancer is present?
LD-4 and LD-5
What type of sample is preferred for LD measurement?
Serum
Why isn’t plasma preferred when LD measurement is done?
May be contaminated with platelets, which contain a high concentration of LD
Does hemolysis affect LD measurement?
Yes, it will falsely elevate the levels due to LD being present in RBCs
Which LD isoenzymes are labile to cold?
LD-4 and LD-5
What is recommended storage for serum used for LD measurement?
Keep at room temp because some of the LD isoenzymes are sensitive to cold
In children, are LD levels normally higher or lower than adults?
5x higher
Measurement of LD depends on what?
Whether the reaction is occurring in a forward or reverse direction
Why is the LD forward reaction the recommended way to measure it?
Lactate is more stable than pyruvate as a substrate, less inhibitory
What is actually measured when LD enzymatic activity is assayed?
As lactate is oxidized to pyruvate, NAD is reduced to NADH. There will be an increase of absorbance at 340 nm.
Why would LD isoenzymes be fractionated?
To determine the organ of involvement in the disease process
Which isoenzyme can be isolated using substrate selectivity with 2-oxybutyrate?
LD-1, due to it containing only H subunits, which 2-oxybutyrate has a greater affinity for
Immunoprecipitation is used to measure all LD isoenzyme fractions except for?
LD-1
Which enzyme group does creatine kinase belong to?
It is a transferase
What reaction does CK catalyze?
Catalyzes the reversible transfer of a phosphate group from ATP to creatine
What is pH optimum for forward CK activity?
9.0
What is the pH optimum for reverse CK activity?
6.8
What ion does CK require to activate the reaction?
Mg
What compounds can act as inhibitors on CK?
(Excess) Mg, Ca, Zn, Cu, sulfhydryl binding reagents, citrate, fluoride, uric acid, excess ADP
What is the molecular structure of CK?
It is a dimer; 2 peptide chain, B and M joined by disulphide bonds
How many CK isoenzymes are there?
3 (CK-1, CK-2, CK-3/CK-BB, CK-MB, CK-MM)
How is CK relatively unstable?
Activity is easily lost when the thiol group in the disulphide bond is broken.
Can the disulphide bond in the CK molecule be restored?
Partial restoration can occur by adding sulfhydryl compounds
What tissue sources is CK-1 (CK-BB) mainly contained in?
Brain and nerve tissue
What tissue sources is CK-2 (CK-MB) mainly contained in?
Heart and skeletal muscle
What tissue sources is CK-3 (CK-MM) mainly contained in?
Heart and skeletal muscle
What are CK isoforms?
Subtypes of the CK isoenzymes
How are CK isoforms created?
Arise from the removal of the terminal lysine on the M peptide chain of the isoenzymes released from the tissues
What enzyme catalyzes the removal of the terminal lysine from the M peptide chain on the CK molecule?
Carboxypeptidase
How many isoforms does CK-3 (CK-MM) have?
3
Which CK-3 isoform has both lysines intact?
MM3
Why doesn’t carboxypeptidase have any effect on the CK-1 (CK-BB) isoenzyme?
The enzyme only has an effect on the M peptide chain. The CK-1 isoenzyme has only B peptide chain.
Which CK-2 (CK-MB) isoform does not have lysine removed?
MB2
Which CK-2 (CK-MB) isoform has one lysine removed?
MB1
How long does it take to remove the lysine from the M peptide chains to form the CK isoforms?
Takes about 10 hours
Which CK-3 isoform has only one of the lysines removed?
MM2
Which CK-3 isoform has both lysines removed?
MM1
