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PURINE & PYRIMIDINE - BIOSYNTHESIS
• New Purines & pyrimidines are formed from amphibolic intermediates,
hence they are classified as non-essential.
• IMP biosynthesis requires folate derivatives & glutamine.
• IMP’s are further converted to AMP & GMP.
PURINE BIOSYNTHESIS
PURINE BIOSYNTHESIS
PYRIMIDINE BIOSYNTHESIS
PYRIMIDINE BIOSYNTHESIS
BREAKDOWN – PURINE & PYRIMIDINE
CELL DISRUPTION, HOMOGENISATION
Cell Disruption:
• 1st step in any analytical process
• Crude mixtures obtained can be used for metabolite uptake studies, enzyme assays
etc
• Investigations on metabolic compartmentalization can be done
• Generally carried out at low temp, 4ºC, to avoid loss of enzyme activity
• Animal tissue - osmotic shock; exposure to alternate freeze/thaw conditions;
enzymatic digestion by a combination of lipases & proteases; exposure to solvents like
toluene
• Plant tissue – Pectinase & cellulose combinations & Microbes – lysozyme
Apparatus - Mortar & pestle; mechanical shaking with abrasives in Mickle shakers,
liquid shearing in blenders; homogenisers with power driven (Potter-Elvejham) pestle
made of pyrex glass, Teflon or leucite can also be used; controlled solid shear with
Hughes Press that generate pressure upto 108 Pa can be used to break plant cells.
HOMOGENISATION
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Sucrose soln is used to provide sufficient osmotic potential to prevent
organelles from swelling or bursting. Can be substituted with sorbitol or
mannitol when it interferes with enzyme assay.
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Mg2+ is important to maintain integrity of nucleus & ribosomes. Alternately,
when membrane proteases need to be inactivated EDTA or EGTA are added
to medium that cause chelation of Mg2+ & Ca2+.
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For many enz the –SH group must be maintained in reduced form, this is done
by addition of 2-mercaptoethanol or dithiothreitol.
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Generally, aqueous medium is used in separating organelles eg. Citrate is
preferred during isolation of nuclei becos it inactivates neutral DNAses.
Sometimes, a non-aqueous medium can also be used eg. Ether-chloroform or
benzene-carbon tetrachloride to prepare chloroplasts, hemosiderin mol from
spleen.[disadvantage- surface of tissue altered; and most enz are inactivated].
CELL FRACTIONATION
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In differential centrifugation, the material is
separated into various fractions by applying
increasing centrifugal field.
Pellet contains sedimented material and
supernatant, the unsedimented.
Separation is determined by the time &
speed of centrifugation and size & density
of particle.
COLORIMETRY
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Identification of a compound is based on the color produced.
Uses colored filters that absorb a limited range of wavelengths called
‘bandwidth’
Standardisation is done by setting the instrument to zero using the blank
A set of standards ranging from lower to higher conc is used to produce a
concentration vs absorbance plot called Beer-Lambert plot. From this the
unknown compound conc is calculated.
Conc = Test absorbance
----------------------------Standard absorbance
The linearity of plot does not continue indefinitely, but becomes saturated after
a point, this is called the Job effect.
In general, the filter should be of a color complimentary to that of the solution
under test.
The colorimeter consists of a light source, filter, cuvette and photosensitive
detector.