Definition: Isozymes may be defined as multiple forms of
the same enzymes having similar, if not identical enzymatic properties due to
amino acid substitutions in their structure or slight differences in the
tertiary or quarternary structures.
How are isozymes generated:
- Multiple alleles at the same locus.
The enzymes produced due to such alleles are known as Allozymes. The number of alleles
present at a particular locus is dependent upon ploidy number of the organism
and its genetic makeup (homozygous or heterozygous). The number of allozymes
present depend upon the number of alleles present. Each allozyme codes for a
different polypeptide chain. If the enzyme is monomeric and the individual is
homozygous, simple band patterns of the allozymes will be seen. However the
complexity of these patterns will increase if the individual is heterozygous
and the enzyme under consideration is a multimeric protein.
- Single or multiple alleles at multiple loci
This is another way in which isozymes may
be generated. Multiple alleles at different loci generally result in formation
of isozymes that may be expressed in different tissues or maybe
compartmentalized in a cell. Such compartmentalization or differential
expression means they can be tightly regulated and may be directed towards
different metabolic functions at different periods of time in different tissues
under slightly varying conditions. Malate dehydrogenase isozymes are the best
example of this.
Also such isozymes may prove to be a useful tool in detecting metabolic anomalies or diseases since under a particular condition only one type of isozyme may be affected while the other is not, leading to a different band pattern being observed as compared to the normal band pattern. Therefore detection of isozymes is an important diagnostic tool. eg: salivary amylase isozymes are detected to confirm whether the person suffers from pancreatic disorders.
However the band patterns generated by such isozymes are
even more complex and hence difficult to interpret.
- Secondary Isozymes
Post translational changes occurring in an
enzyme structure may also lead to isozyme formation; such isozymes being termed
as secondary isozymes. These enzymes are first synthesized normally(primary
enzymes) which then may undergo changes in vivo or in vitro.
In vivo changes include secondary steps
like methylation, acetylation, phosphorylation, sialation, cleavage by
proteolytic enzymes, loss of amide groups and addition of carbohydrate side
chains to reactive residues.
Isozymes may be generated in vitro because
of storage, improper handling of the specimens or degradative reactions.
Conformational isozymes, having same
chemical structure but different 3 dimensional conformations, are also included
under secondary isozymes. These are believed to be interconvertible by chemical
means but till date no definite examples have been found.
Detection of isozymes can be done by methods like zone electrophoresis,
gel filtration chromatography, PAGE to name a few. All these techniques are
used for separation of the isozymes. Detection involves activity staining or
zymogen staining procedures. The stains used are specific for specific enzymes;
some maybe enzyme specific while some maybe group specific. However only active
enzymes are detected by these techniques. This may be perceived as a limitation
of the techniques. Another disadvantage is that Only isozymes having a
considerable difference in charge or conformation can be detected. However,
this may not always be the case and hence many of the isozymes may remain
undetected.
Isozyme studies have found use in detecting
and differentiating unknown organisms and plant pathogens . They can be used in
detecting the homozygosity or heterozygosity of individuals as well as
classifying them in the appropriate taxonomic group.
References:
Isozymes: Methods and Applications; J.
A. Micales and M. R. Bonde
Isozymes; D.A.Hopkinson; journal of
clinical pathology, vol 8 1974 pg 122-127
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