<p> Cell Fractionation </p><p>Summary & Revision</p><p>Experimental animals</p><p>Rats , mice , rabbits , guinea pigs</p><p>Kreb's Cycle</p><p>(Tricarboxylic acid cycle ( TCA</p><p>Citric acid Cycle</p><p>ATP = Biological battery</p><p>(NAD : nicotineamide adenine dinucleotide ( oxidized form</p><p>( reduced form ) </p><p>: NADP</p><p>Energy charges</p><p>↑ ATP</p><p>AMP + 2 P = ATP</p><p>ADP + P = ATP</p><p>ADP , AMP</p><p>NADH , NAD</p><p>1  ما المقصود ؟؟ </p><p>هل يمكن عزل جميع عضيات الخلية في صورة سليمة ؟؟؟</p><p>عزل النواة - التحقق من عزل النواة </p><p>عزل الميتوكوندريا - التحقق </p><p>عزل الميكروسومات و الليسوسومات - التحقق </p><p>عزل السيتوسول – التحقق </p><p>تقنية الطرد المركزي - النواع – الخصائص </p><p>النزيمات - فكرة عامة - كيفية تقديرها – نشاطية النزيم </p><p>الحسابات و النتائج </p><p>M – 0.34 M Sucrose solution 0.25</p><p>M.W = 342</p><p> ml 100</p><p> g = 0.25 * 342 * 100 / 1000</p><p> g / 100 ml 8.5</p><p>For osmotic pressure</p><p>2 MCQ : Multiple Choice Questions</p><p>Sucrose </p><p>Carbohydrate </p><p>Disaccharide </p><p>Table sugar </p><p>Glucose + Fructose </p><p>C6 H12 O6 72 + 12 + 96 = 180 </p><p>Isomers </p><p>3 Crude DNA </p><p>Diphenyl amine R. ( R. = Reagent )</p><p>DNA ------Deoxyribose ------(HCl) _____ Furfural derivative </p><p>( diphenyle amine ) ______Bluish color </p><p>Standard DNA ( Calf thymus ) </p><p>Crude RNA </p><p>Orcinol ------ribose ------(HCl) _____ Furfural derivative </p><p>( orcinol ) ______Blue – green color </p><p>Confirmatory Tests </p><p>Tm : melting temperature </p><p>Nitrogenous bases A , C , G , T , U </p><p>260 – 280 nm ( U.V. region ) ultra violet </p><p>U.V. absorption method for Nucleic acids ( for pure samples ) </p><p>تنقية Purification</p><p>Nucleoproteins ( conjugated protein) </p><p>Histones </p><p>1% E 1Cm for DNA at 260 nm = 200 ( Calf Thymus ) </p><p>1g / 100 ml </p><p>1000 mg / 100 ml </p><p>4 10 mg / 1 ml ____ 200 </p><p>1mg / 1ml ______20 </p><p>0.1 mg / 1 ml ______2.0 </p><p>0.01 mg / 1 ml ______0.20 </p><p>1% E 1Cm for RNA at 260 nm = 250 ( yeast ) </p><p>Results : </p><p>? mg DNA ( or RNA) / g liver dry tissue </p><p>أنواع كروماتوجرافيا العمود Column chromatography </p><p>( .Gel filtration ( based on mol.wt</p><p>( Ion exchange chromatography ( charge</p><p>Affinity chromatography</p><p>5 Kreb's cycle</p><p>.β Oxidation of f.as</p><p>Oxidative phosphorylation</p><p>( ETC ( electron transport chain</p><p>IDH isocitrate dehyrogenase</p><p>Microsomes</p><p>( Ultracentrifuge ( 75,000 r.p.m</p><p>Beckmann</p><p>(Mixed function oxidases ( MFO</p><p> eg : aldehyde oxidase , xanthine oxidase , Cyt P450</p><p>Cytosol</p><p>( High speed centrifuge ( 15000 – 18,000 rpm</p><p>( Sorvall ( American company</p><p>6 Glycolysis</p><p>Glucose ______pyruvate</p><p>Pyruvate + NADH ______lactate + NAD</p><p>LDH : lactate dehydrogenase </p><p>RCF : Relative centrifugal force</p><p> xg = ??? rpm 45,000</p><p>E , S , P , PH , T</p><p>S : Substrate</p><p>E : Enzyme</p><p>T:Temperature</p><p>In-vitro</p><p>In-vivo</p><p>Enzyme assay</p><p>Amylase</p><p>Starch ( MW 1.5 * 10 6 ) ______Dextrins ______Disaccharides ( maltose & isomaltose ) ______Glucose ( ( reducing sugar</p><p>7 ( C6H12O6 ( 72 + 12 + 64 = 180</p><p>µ mole produced / min / ml E = U / ml U : unit</p><p>DNS 3,5</p><p>Dinitrosalicylate , yellow – orange in color ( color R for reducing 3,5 ( sugars</p><p>0504677397</p><p>The reducing sugar will reduce NO2 group in position 3 to NH2</p><p>The color will be changed from yellow orange into red</p><p>DNS has two functions </p><p>To stop the enzymatic reaction ( DNS is prepared in 2M Na OH ) </p><p>Will react with glucose produced </p><p>Cytology </p><p>Eukaryotes </p><p>Organelles </p><p>Plasma membrane </p><p>Nucleus </p><p>Cytoplasm = cytosol </p><p>Mitochonderia</p><p>8 Ribosomes </p><p>Endoplasmic reticulum </p><p>Golgi apparatus </p><p>Nucleolus </p><p>Rat liver ( 1 g ) </p><p>0.25 M sucrose </p><p>Homogenization ( Homogenizer ) 1:5</p><p>Centrifugation ( Centrifuge ) </p><p>1. Bench- Top centrifuge ( up to 6000 rpm ) rpm : round per minute </p><p>2. High speed centrifuge ( up to 20000 rpm ) </p><p>Sorvall : American company </p><p>3. Ultra centrifuge ( up to 100000 rpm ) </p><p>Physiological Saline = 0.9% NaCl </p><p>9 Dehydrogenases enzymes e.g . isocitrate dehyrogenase alpha – ketoglutarate dehydrogenase </p><p>Oxidoreductases E.C.1</p><p>Enzyme Commission pyruvate dehydogenase complex </p><p>10 Coenzymes </p><p>NAD , NADP , FAD , NADH , NADPH , FADH 2 </p><p>A = KCL </p><p>C = A / KL </p><p>C = 0.055 * 10 6 µ mol * Cm / 6.22 * 10 3 L * 1Cm </p><p>C = 0.055 * 10 6 µ mole * Cm / 6.22 * 10 3 103 ml * 1 Cm </p><p>No. of µmoles produced of NADH / min / vol. added of mitochondrial fraction </p><p>U/ml </p><p>U / g wet tissue </p><p>1M 3 -1 -1 E 1Cm for NADH ( NADPH ) at 340 nm = 6.22 * 10 L mol Cm </p><p>Zero time A = 0.726 </p><p>1 min. = 0.772 </p><p>2 min = 0826 </p><p>3 min = 0.872 </p><p>4 min = 0.934 </p><p>5 min = 1.00 </p><p>11 0.046 </p><p>0.054</p><p>0.046</p><p>0.062 </p><p>0.066 </p><p>∆ A / min = 0.055 </p><p>Some Lysosmal enzymes </p><p>12 Proteases ( for protein degradation ) </p><p>Nucleases ( eg, ribonucleases , deoxyribonucleases )</p><p>Lipases ( for lipid degradation ) </p><p>Galactosidases ( α – galactosidases , β- galactosidases ) </p><p>Phosphatases ( for phosphate removal ) </p><p>Lysosomal storage diseases ( LSD ) </p><p>Cytosol fraction </p><p>18,000 rpm ( High speed centrifuge ) </p><p>Marker : lactate dehydrogense LDH </p><p>Sod. Pyruvate + NADH + H + → lactate + NAD + </p><p>- + + - + CH3 C=O COO Na + NADH + H → CH3 CHOH COO + NAD </p><p>P → L </p><p>L → P </p><p>A = KCL </p><p>13 C = A / KL </p><p>C = 0.036 * 10 6 µ mol * Cm / 6.22 * 10 3 L * 1Cm </p><p>C = 0.036 * 10 6 µ mole * Cm / 6.22 * 10 3 103 ml * 1 Cm </p><p>No. of µmoles disappeared of NADH / min / vol. added of cytosolic fraction </p><p>U/ml </p><p>1 : 5 ( 1g / 5 ml )</p><p>1ml = 0.2 g </p><p>U / g wet tissue </p><p>1M 3 -1 -1 E 1Cm for NADH ( NADPH ) at 340 nm = 6.22 * 10 L mol Cm </p><p>Zero time A = 0.726 </p><p>1 min. = 0.682 </p><p>2 min = 0.640 </p><p>3 min = 0.60</p><p>4 min = 0.31 </p><p>5 min = 0.12 </p><p>0.044</p><p>14 0.042</p><p>0.040</p><p>0.029</p><p>0.019</p><p>GOUT ( disorder of uric acid metabolism ) </p><p>Cofactor </p><p>1. Coenzyme </p><p>2. Metal ion ( Mg , Zn , Ca …… etc ) </p><p>15 3. Prosthetic group </p><p>Hydrophilic </p><p>Hydrophobic </p><p>GluT2 </p><p>Hydrology </p><p>Microsomal & lysosomal fraction </p><p>Ultra centrifuge ( 75000 rpm ) </p><p>Marker or indicator </p><p>Microsomal fraction </p><p>1. Glucose -6- phosphatase ( G6Pase ) </p><p>G6p → Glucose + Phosphate ( Pi ) </p><p>+6 Phosphate ( Pi ) +Ammonium molybdate → Phosphomolybdate ( Mo ) </p><p>+3 + Reducing agent ( Vit.C : ascorbic acid ) → ( Mo ) Mo2O3 </p><p>Molybdenum blue </p><p>Specificity </p><p>Mo : molybdenum </p><p>16 2. MFO : Mixed Function Oxidases ( drug metabolism ) </p><p>Xenobiotics </p><p>Aldehyde oxidase </p><p>Xanthine oxidase </p><p>Lysosomal fraction </p><p>Indicator : Acid phosphatase ( ACP) </p><p>17 P- nitrophenyl phosphate → P-nitrophenol + Pi </p><p>Yellow in color </p><p>Λmax = 400 – 405 nm </p><p>Increase in A </p><p>Extinction coefficient = 18.7 </p><p>Prostate cancer ( clinical application ) </p><p>18</p>