Transcript Slide 1
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.