CC LEC 2 - Enzymes II Flashcards
128,000 Daltons
Interconversion of lactic acid and
pyruvic acid
LD - Lactate Dehydrogenase
Atypical LD
LD-6: ??
alcohol dehydrogenase
Atypical LD Migrates cathodal to LD-5 Present in patients with arteriosclerotic cardiovascular failure Grave prognosis, signifies impending death
alcohol dehydrogenase
Atypical LD
LD complexed with IgA or IgG, migrates between LD-3 and LD-4
Macro-LD:
– to measure LD-1(HHHH) because H-units have great affinity
*a-Hydroxybutyrate dehydrogenase activity
Methods for LD forward reaction - mixture of phenazine methosulfate and nitroblue - tetrazolium reacts with NADH producing blue purple color - pH8.3–8.9
- Wacker method
Methods for LD – reverse reaction - NADH is cosubstrate, consumed during reaction
- pH7.1–7.6
Wroblewski La Due method
Methods for LD - 3 times faster, smaller sample, shorter reaction time - Susceptible to substrate exhaustion and loss of linearity
Wroblewski La Due method
Transfer of amino group between aspartic acid and a-ketoacids
Aspartate Aminotransferase
ast coenz
Pyridoxal phosphate is coenzyme
ast isoenz
predominant form in serum
Cytoplasmic Isoenzyme –
ast isoenz
increased during cell necrosis
Mitochondrial Isoenzyme
AST Method – coupled enzymatic reaction - Malate dehydrogenase is indicator enzyme - Decrease in absorbance at 340nm - Optimal pH 7.3-7.8
Karmen method
AST Method
– ketoacids react with 2,4 – DNPH to form ketoacid hydrazines
- Product is intense brown color measured at 505nm
Reitman-Frankel Method
- Catalyzes transfer of amino groups from alanine to a-ketoglutarate
Alanine Aminotransferase
Liver specific enzyme
Alanine Aminotransferase
Not elevated in heart disease unless accompanied with liver disease
Alanine Aminotransferase
Tends to be higher and lasts longer than AST in acute inflammatory conditions of the liver
Alanine Aminotransferase
Requires Mg2+ and Mn2+ as activators
Alkaline Phosphatase
class 3 enz
ALP
Hydrolysis of various phosphomonoesters; making it nonspecific in alk
ALP
ALP isoenz Increased in B and O secretors
Intestinal ALP
Abnormal ALP isoenz
Regan
Nagao
Abnormal ALP
Variant of Regan
- Detected in metastatic carcinoma of pleural surfaces and in adenocarcinoma of pancreas and bile duct
nagao
Abnormal ALP
Highest in ovarian and gynecologic cancers
- Used for monitoring treatment because it disappears upon successful therapy
regan
ALP ISOENZ (3) are most heat stable and resist denaturation at 65C for 30 minutes
Placental, Regan, and Nagao
inhibits intestinal (75%) and placental (80%) ALP ISOENZ
Phenylalanine
Nagao is also inhibited by __
L-leucine
inactivates bone(90%0 more than liver(60%) ALP
Urea(2M)
Hydrolysis of various phosphomonoesters at acidic pH (5.0)
ACP
inhibits prostatic ACP
Tartrate
inhibits non-prostatic ACP
Copper
__ fastest towards anode; __closer to the cathode
pACP fastest towards anode; eACP closer to the cathode
Prostatic cancer stage
Small islands of cancer cells in prostate
Normal Population
1
Prostatic cancer stage
Presence of small nodules in prostate
70-90% live for 5 years
2
Prostatic cancer stage
Presence of nodes in the prostate and spread around the pelvic area
40-70% live for 5 years
3
Prostatic cancer stage
Nodules inside and outside prostate, metastasizing in bones and presence of lymph nodes
15-20% live for 5 years
4
First seen in alcohol intake
GGT
Transfers gamma glutamyl from gamma glutamyl peptides to Amino Acids, H2O, and other small peptides
GGT
serves as the gamma glutamyl donor in most biological systems
Glutathione
is present in large extent in smooth ER and is subjected to hepatic microsomal induction
GGT
Used as a differentiating source of ALP elevation
GGT
Mtd of meas for GGT
Szasz
MW of amylase
50 000 - 55 000 daltons
Breakdown of starch & glycogen with the products
AMS
Requires calcium & chloride ions for
activation
AMS
Tx sources of AMS
Acinar cells of pancreas
Salivary glands
long, unbranched chain of
glucose molecules, linked by α, 1-4
glycosidic bonds
Amylose
branched chain
polysaccharide with α, 1-6 linkages
at the branched points
Amylopectin
similar to amylopectin but more highly branched
Glycogen
ISOENZ of AMS
- ## From pancreatic tissue
P-type (P1, P2, P3)
ISOENZ of AMS
Predominates in urine
P-type (P1, P2, P3)
ISOENZ of AMS
markedly elevated in acute
pancreatitis & renal failure
P3
ISOENZ of AMS
From salivary gland tissues, fallopian
tubes, lungs
S-type (S1, S2, S3)
ISOENZ of AMS
Inhibited by wheat germ lectin
S-type (S1, S2, S3)
ISOENZ of AMS
Migrates faster than P-type
S-type (S1, S2, S3)
ISOENZ of AMS
Predominates in serum (2/3 of AMS
activity in serum)
S-type (S1, S2, S3)
Most commonly observed AMS ISOENZ fractions
P2, S1, S2
AMS Method
Measures the disappearance in initial dark blue color of starch-iodine complex
- Fast disappearance = high AMS activity
Amyloclastic/Iodometric Method
AMS Method
Reduction test, reference method
- Starch substrate hydrolyzed by AMS
to its constituent carbohydrate molecule that has reducing properties like sugars
- Amount of reducing sugar = AMS activity
Saccharogenic Method
AMS Method
Employs a starch substrate with a chromogenic dye that forms an insoluble dye-substrate complex
- With AMS, a smaller soluble fragment of the dye-substrate is formed
- ↑ color intensity of soluble dye- substrate complex = ↑ AMS activity
Chromolytic/Chromogen Labelled Substrate Method (Colorimetric)
AMS Method
- Replaced the previous methods in the lab
- Measures the change in absorbance of NAD+ at 340 nm at pH 6.9
- Substrate: maltotetrase/maltopentoase
Coupled Enzyme Reaction/Continuous Monitoring Method
of mg glucose in 30 minutes at 37°C at specific assay conditions
Somogyi units:
