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Metabolic product and intermediate form during biosynthesis is
controlled by various enzymes.
Types of Metabolite :
• Oxidation
• Reduction
• condensation
• Amination
• Methylation
• Cyclization etc.
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TECHANIQUES USED FOR INVESTIGATION OF BIOSYNTHETIC
PATHWAYS
Tracer techniques
Use of Isolated organs or Tissues
Grafting method
Use of mutant strains
Enzymatic studies
1. TRACER TECHANIQUES:
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PREPARATION OF LABELED COMPOUND:
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Tritium labeling is effected by catalytic exchange (Pt catalyst) in
aqueous media by hydrogenation of unsaturated compounds with
tritium gas.
ROOT FEEDING:
The plant in which roots are the biosynthetic sites, this method is preferred.
E.g.Tobacco
In this type of experiment the plant are cultivated hydroponically to avoid
microbial contamination.
In this method substrate can be administered through the cut ends of stem
immersed in a solution. For Latex containing plant this method is not
suitable.
DIRECT IJECTION:
This method is applicable to the plant with hollow stem. E.g. Umbelliferae
and capsule bearing plant (opium poppy)
FLOATING METHOD:
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When small amount of material is available, floating method is used. In this
method, leaf disc or chopped leaves are floating on the substrate solution.
This technique is also used in conjugation with vacuum infiltration to
remove gases.
SPRAY TECHNIQUE:
In this method compound have been absorbed after being sprayed on leaves
in aqueous solution. E.g. Steroids.
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These entire instruments characterize the nature of radiation. Basically it
depends upon the conversion of kinetic energy of particle into fleeting pulse
of light as a result of its penetration into a suitable luminescent medium.
For stable isotopes,
• Mass spectroscopy- gives molecular peaks depending on mass/charge
ratio.
• NMR- gives nature of carbon and proton.
Methods in Tracer Techniques
Competitive feeding:
A C
B1
Competitive feeding can distinguish whether B or B1 is the normal
intermediate in the formation of C from A.
E.g. Biosynthesis of Hemlock alkaloids (Coniine and conhydrine)
Sequential Analysis:
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obtained the sequence in which various related compounds becomes labeled
from the results some biosynthetic route may be accepted and others
rejected.
• 14CO2 and sequential analysis has been very successfully used in the
elucidation of the path of carbon in photosynthesis.
• Determination of sequential formation of opium and tobacco
alkaloids.
2. USE OF ISOLATED CELL, TISSUE AND ORGAN:
3. ENZYMATIC STUDIES:
4. GRAFTING METHOD:
Grafted plant is especially use in finding the main site of primary as well as
secondary metabolite formation.
Grafting techniques have been useful in providing principle sites:
For capsicin in developing capsicum plants
Aerial plants parts of cannabis for resin
Roots of nicotiana tobacum for nicotine
Leaves ofnicotiana glauca for anabasine
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5. Mutant strand:
Physical: Radiation
Chemical: Colchicin.
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