Isoenzyme biochemistry mbbs

1. ISOENZYMES

2. • Physically distinct form of enzyme
• catalyzing same reaction
• Have multiple molecular forms
• Synthesized from various tissues
Eg salivary and pancreatic
amylase

3. • If all subunits same – protein is
homomultimer – single gene
• If subunits different – protein is
heteromultimer – different gene

4. • When they are products of different
gene – true isoenzymes
• Some cases – all different forms
present in same individual
LDH – 5 forms – all present in all
individuals

5. • Some cases – only one form will be
present in one individual
G6PD – has 400 distinct forms
• Allozymes ---Multiple forms of an
enzyme encoded by different alleles in
one gene locus

6. • 1- Electrophoresis
Agar gel or Polyacrylamide gel electrophoresis
LDH, CK, ALP --- has different mobility
• 2- Heat stability – denatured at different temp
• 3- Inhibitor – one form can be sensitive to particular
inhibitor
• 4- Km value or Substrate Specificity –
glucokinase has high Km for glucose
hexokinase has low Km for glucose
Identification

7. • 5- Co factor requirement –
mitochondrial isocitrate dehydrogenase requires NAD+
cytoplasmic isocitrate dehydrogenase requires NADP+
• 6- Tissue localization –
HHHH form of LDH --- In heart
MMMM form of LDH --- in muscle
• 7- By ELISA, Protein purification

8. Creatinine kinase
or creatinine phosphokinase
creatinine + ATP creatinine-P +
ADP
• Normal reference range -- 15 – 100 U/L
(less in females)

9. • Dimer of 2 units – M (muscle) & B (Brain)
BB (CK1) brain most mobile 1%
MB (CK2) heart 5%
MM (CK3) muscle least mobile 80%
• Total CK- muscular dystrophies
• CK-MB isoenzyme- diagnostic marker of MI

10. • Increase levels seen in
-- MI within 3-6 hrs - detect early cases
-- Muscular dystrophy
-- Crush injury & Fracture
-- Cerebrovascular Accidents(Stroke)

11. Lactate dehydrogenase
catalyze conversion of pyruvate to lactate
pyruvic acid + NADH + H+ lactic acid + NAD+
• Normal reference range in serum --- 100 – 200 U/L
• LDH in RBC – 100 times more – compared to plasma
• Tetramer of 4 subunits LDH 1 HHHH Heart
LDH 2 HHHM RBC
LDH 3 HHMM Brain
LDH 4 HMMM Liver
LDH 5 MMMM Muscle

12. • Normally LDH-2 is more than LDH-1
• Pattern reversed in MI
• Flipped pattern
• LDH is non specific
• LDH has no diagnostic value

13. • Separated by cellulose acetate / agarose
electrophoresis
LDH 1 more +ve charge --- moves fastest
LDH 5 more - ve charge --- moves slowest
• In MI --- LDH 1 increases 5-10 times with in 12 hrs
• Levels increase in --- hemolytic anemia, muscle
dystrophy, hepatocellular damage, leukemia

14. Alkaline phosphatase
-- optimum pH 9-10, contains Zn,
activated by Mg
-- produce by osteoblast of bone – for
calcification
• Normal reference range -- 40 – 125 U/L
( more in children)

15. • Moderate increase 2-3 times – hepatic disease
infective hepatitis
alcoholic hepatitis

16. • high level - 10-12 times
Extrahepatic /obstructive jaundice
Gall bladder stone & carcinoma
Carcinoma pancreas
Intra hepatic -- viral infection

17. • Very high level -- up to 25 times
bone disease -- Paget’s disease,
Osteomalacia, Rickets
hyperparathyroidism

18. • Isoenzymes – 6 forms
ALP1 – increase in obstructive jaundice
ALP1 & 2 – present in liver
pre β ALP – in bone
Placental ALP
γ ALP – intestine (Ulcerative colitis
igG bound ALP

19. • G6PD
Glucose 6 phosphate dehydrogenase
Dimer with identical subunit
Produces NADPH
• H2O2 – forms inside RBC – prevented by NADPH
and Glutathione
• G6PD decrease in – drug induced hemolytic anemia
– met hemoglobinemia