Azhar University,

Damietta Faculty of medicine,

Biochemistry and Molecular Biology

department.

Medical Research on:

Isoenzymes
BY:
61-Eslam Hosiny Abdelrehim.

62-Eslam Khaled Mohamed.

63-Eslam Rafeek Mohamed.

64-Eslam Saied Ahmed.

65-Eslam Mohamed Ibrahim.

Under supervision of:

Prof Dr/ Mahmoud Abdelghany Elsayed.

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Introduction:

Isoenzymes are multiple forms of the enzyme that have the

same catalytic activity, but differ in electrophoretic mobility,
physical properties and liability to enzymes inhibitors.

Contents:
1-Some details about isoenzymes (properties, source etc.).

2-Examples of isoenzymes.

3-Lactate Dehydrogenase (LDH).

4-Creatine Kinase (CK).

5-Alkaline Phosphatase (AP).

6-Summary.

Properties:
1-They are series of different proteins.

2-They have the same chemical properties but differ in

physical properties.

3-They have electrophoretic mobility.

4-They can be demonstrated.

Methods of demonstration of isoenzymes:

The most popular method is by using of specific staining for

enzyme activity it is then possible to demonstrate the
positions taken up by the individual isoenzymes. (Banner,
1989)

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Sources of isoenzymes:
1-They may be produced by the same gene but the protomers
undergo different post-translation modifications in different
organs (especially Golgi complex).

2-They may be produced by many genes; each gene produces

one protomer.

Medical Importance of Isoenzymes:

Isoenzymes are not only important for diagnosis but also
indicate the diseased organ.

Major examples of isoenzymes:

1-Lactate Dehydrogenase (LDH).

2-Creatine Kinase (CK).

3-Alkaline Phosphatase (AP).

Lactate Dehydrogenase (LDH).

Lactate Dehydrogenase (LDH) is the enzyme that catalyzes
the removal of 2 hydrogen atoms from lactic acid to form
pyruvic acid.

N.B:The enzyme in human beings and in a variety of other

animals (Banner, 1989).

Structure:
LDH enzyme is a tetramer formed of 4 protein subunits; each
subunit is called (protomer).
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The protomers of LDH are 2 types, H and M. So, LDH exists in
5 isoenzyme forms in the body:

1-LDH1 (HHHH) which increases in myocardial infarction.

2-LDH2 (HHHM) which increases in myocardial infarction.

3-LDH3 (HHMM) which increases in leukemia.

4-LDH4 (HMMM) which increases in viral hepatitis and muscle

diseases.

5-LDH5 (MMMM) which increases in viral hepatitis and muscle

diseases.

N.Bs:

1-The LDH1 isoenzyme tends to predominate in those tissues

which respire aerobically, whereas the LDH5 tends to
predominate in those tissues which respire more or less
anaerobically.

2-The PH of LDH electrophoretic mobility is 8.

3-LDH1 is the fastest enzyme in electrophoretic mobility, while

LDH5 is the slowest.(Banner, 1989).

4- "LDH1 (HHHH) tends to associate with the mitochondrial

membrane and LDH5 (MMMM) with the nucleus."(Banner,
1989).

Function:
LDH isoenzymes are clinically important to differentiate
between heart, liver and blood diseases.

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Example:
LDH1 (HHHH) is one of cardiac markers which increase in
myocardial infarction, reaches its peak after 48-72 hours, and
backs to its normal after 10-14 days.

Creatine Kinase (CK).

Introduction:

"Creatine kinase (CK) is a central controller of cellular energy

homeostasis. By reversible interconversion of creatine into
phosphocreatine, CK builds up a large pool of rapidly diffusing
phosphocreatine for temporal and spatial buffering of ATP
levels. Thus, CK plays a particularly important role in tissues
with large and fluctuating energy demands like muscle and
brain." (Schlattner, Tokarska-Schlattner, & Wallimann, 2006).

The enzyme creatine kinase (CK), catalyzing the reversible

transfer of the N-phosphoryl group from phosphocreatine
(PCr) to ADP to regenerate ATP, plays a key role in the energy
homeostasis of cells with intermittently high, fluctuating
energy requirements, e.g., in skeletal and cardiac muscle,
neurons,
photoreceptors, spermatozoa and electrocytes.
Cytosolic CK isoenzyme(s) (MM-, MB- and BB-CK) are always
co expressed in a tissue-specific fashion together with a
mitochondrial isoform.(Wallimann et al., 1998)

Structure:
CK enzyme is a dimmer formed of 2 protein subunits, B and
M. So, Creatine Kinase has 3 isoenzymes:

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1-CK1 which formed of BB and increases in brain tumors.

2-CK2 which formed of MB and increases in heart diseases.

3-CK3 which formed of MM and increase in skeletal muscle

diseases.

Function:
1-Creatine Kinase isoenzymes are used to differentiate
between brain, heart and skeletal muscles diseases.

2-"Cytosolic CKs are in close conjunction with Ca 2+ pumps,

play a crucial role for the energetics of Ca 2+
homeostasis."(Wallimann et al., 1998)

3-"Mitochondrial Creatine Kinase (MI-CK), a cuboidal shaped

octamer with a central channel, binds and crosslinks
mitochondrial membranes and forms a functionally coupled
micro compartment with porin and adenine nucleotide
translocase for vectorial export of PCr into the
cytosol."(Wallimann et al., 1998).

Example:
Cardiac Creatine Kinase (CK-MB) is one of most specific
cardiac markers which increases in myocardial infarction,
reaches its peak after 10-24 hours, and backs to its normal
after 24-72 hours. However, it can't be used for diagnosis of
later acute MI.

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 Alkaline Phosphatase (AP).

Introduction:
"The Alkaline Phosphatase family catalyzes the hydrolysis of
phosphomonoesters to produce inorganic phosphate and an
alcohol from a variety of organic compounds as nucleosides
(5 -tri-, -di- and -mono- phosphates), inorganic
pyrophosphate, pyridoxal-5-phosphate and phosphorylated
proteins."(Deracinois, Lenfant, & Dehouck, 2015).

Structure:
Six bands with alkaline phosphatase (ALP) activity can be
detected by electrophoresis using cellulose acetate
membrane:

1-ALP1: high molecular weight ALP.

2-ALP2: hepatic ALP.

3-ALP3: bone ALP.

4-ALP4: placental ALP.

5-ALP5: intestinal ALP

6-ALP6: igG bound ALP.

N.B:

1-Identification or quantitative measurement of each isozyme

is possible by using heat inactivation, various inhibitors, gel
filtration, immunoassay, etc.

2-"The number of observations of an unexpected elevation of

serum alkaline phosphatase has been increased by the
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practice of many laboratories of organizing their
investigations into multi-test profiles, which almost invariably
include measurement of serum alkaline phosphatase."(Moss,
1983).

Site:
Most tissues contain Alkaline Phosphatase, but AP activity is
important in kidney, liver, bone, intestine, placenta and
reticuloendothelial tissue, especially in the endothelial cells of
brain.(Deracinois et al., 2015).

Function:
1- The enzyme alkaline phosphatase is an important serum
analyte and its elevation in serum is correlated with the
pressure of bone, liver, and other diseases.

2- Bone alkaline phosphatase (BALP) is one of the most

frequently used biochemical markers of bone formation.

Summary:
Isoenzymes are multiple forms of enzymes that have the
same catalytic activity and differ in physical
properties.

Isoenzymes can be produced by one gene or more.

Isoenzymes have many examples as:

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1-LDH which consists of 5 subunits and used as a
marker for diagnosis of heart, blood, and liver
diseases.

2-CK which consists of 3 subunits and used as a

marker for diagnosis of heart, brain, and muscle
diseases.

3-ALP which consists of 6 subunits and increase in

bone and liver diseases.

Sources:
1-Banner, F. T. (1989). The nature of, (3), 171179.

2-Deracinois, B., Lenfant, A., & Dehouck, M. (2015). Neuronal

Tissue-Nonspecific Alkaline Phosphatase (TNAP), 76, 125
151. https://doi.org/10.1007/978-94-017-7197-9

3-Moss, D. W. (1983). Clinical biochemistry of alkaline

phosphatase. Cell Biochemistry and Function, 1(2), 7074.

4-Schlattner, U., Tokarska-Schlattner, M., & Wallimann, T.

(2006). Mitochondrial creatine kinase in human health and
disease. Biochimica et Biophysica Acta (BBA) - Molecular
Basis of Disease, 1762(2), 164180.
https://doi.org/10.1016/j.bbadis.2005.09.004

5-Wallimann, T., Dolder, M., Schlattner, U., Eder, M.,

Hornemann, T., OGorman, E., Brdiczka, D. (1998).
Some new aspects of creatine kinase (CK):
compartmentation, structure, function and regulation for

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 cellular and mitochondrial bioenergetics and physiology.
BioFactors (Oxford, England), 8(34), 229234.
https://doi.org/10.1002/biof.5520080310

6-Pubmed.

7-Practical book, medical biochemistry department, Azhar

university.

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