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• 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.

• 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+.

• For many enz the –SH group must be maintained in reduced form, this is done

by addition of 2-mercaptoethanol or dithiothreitol.

• 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

• 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

• 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.