Cell Fractionation
Summary & Revision
Experimental animals
Rats , mice , rabbits , guinea pigs
Kreb's Cycle
Tricarboxylic acid cycle ( TCA)
Citric acid Cycle
ATP = Biological battery
NAD : nicotineamide adenine dinucleotide ( oxidized form)
( reduced form )
NADP :
Energy charges
ATP ↑
AMP + 2 P = ATP
ADP + P = ATP
ADP , AMP
NADH , NAD
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‫‪ ‬ما المقصود ؟؟‬
‫هل يمكن عزل جميع عضيات الخلية في صورة سليمة ؟؟؟‬
‫عزل النواة ‪ -‬التحقق من عزل النواة‬
‫عزل الميتوكوندريا ‪ -‬التحقق‬
‫عزل الميكروسومات و الاليسوسومات ‪ -‬التحقق‬
‫عزل السيتوسول – التحقق‬
‫تقنية الطرد المركزي ‪ -‬األنواع – الخصائص‬
‫األنزيمات ‪ -‬فكرة عامة ‪ -‬كيفية تقديرها – نشاطية األنزيم‬
‫الحسابات و النتائج‬
‫‪0.25 M – 0.34 M Sucrose solution‬‬
‫‪M.W = 342‬‬
‫‪100 ml‬‬
‫‪g = 0.25 * 342 * 100 / 1000‬‬
‫‪8.5 g / 100 ml‬‬
‫‪For osmotic pressure‬‬
‫‪2‬‬
MCQ : Multiple Choice Questions
Sucrose
Carbohydrate
Disaccharide
Table sugar
Glucose + Fructose
C6 H12 O6 72 + 12 + 96 = 180
Isomers
3
Crude DNA
Diphenyl amine R. ( R. = Reagent )
DNA -------- Deoxyribose ----------(HCl) _____ Furfural derivative
( diphenyle amine ) ____________ Bluish color
Standard DNA ( Calf thymus )
Crude RNA
Orcinol -------- ribose ----------(HCl) _____ Furfural derivative
( orcinol ) ____________ Blue – green color
Confirmatory Tests
Tm : melting temperature
Nitrogenous bases A , C , G , T , U
260 – 280 nm ( U.V. region ) ultra violet
U.V. absorption method for Nucleic acids ( for pure samples )
Purification
‫تنقية‬
Nucleoproteins ( conjugated protein)
Histones
E 1%1Cm for DNA at 260 nm = 200
( Calf Thymus )
1g / 100 ml
1000 mg / 100 ml
4
10 mg / 1 ml ____ 200
1mg / 1ml ______ 20
0.1 mg / 1 ml _______ 2.0
0.01 mg / 1 ml ______ 0.20
E 1%1Cm for RNA at 260 nm = 250 ( yeast )
Results :
? mg DNA ( or RNA) / g liver dry tissue
Column chromatography ‫أنواع كروماتوجرافيا العمود‬
Gel filtration ( based on mol.wt. )
Ion exchange chromatography ( charge )
Affinity chromatography
5
Kreb's cycle
β Oxidation of f.as.
Oxidative phosphorylation
ETC ( electron transport chain )
IDH isocitrate dehyrogenase
Microsomes
Ultracentrifuge ( 75,000 r.p.m )
Beckmann
Mixed function oxidases ( MFO)
eg : aldehyde oxidase , xanthine oxidase , Cyt P450
Cytosol
High speed centrifuge ( 15000 – 18,000 rpm )
Sorvall ( American company )
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Glycolysis
Glucose __________________ pyruvate
Pyruvate + NADH _______________ lactate + NAD
LDH : lactate dehydrogenase
RCF : Relative centrifugal force
45,000 xg = ??? rpm
E , S , P , PH , T
S : Substrate
E : Enzyme
T:Temperature
In-vitro
In-vivo
Enzyme assay
Amylase
Starch ( MW 1.5 * 10 6 ) ________ Dextrins __________
Disaccharides ( maltose & isomaltose ) _____________ Glucose (
reducing sugar )
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C6H12O6 ( 72 + 12 + 64 = 180 )
µ mole produced / min / ml E = U / ml
U : unit
3,5 DNS
3,5 Dinitrosalicylate , yellow – orange in color ( color R for reducing
sugars )
0504677397
The reducing sugar will reduce NO2 group in position 3 to NH2
The color will be changed from yellow orange into red
DNS has two functions
To stop the enzymatic reaction ( DNS is prepared in 2M Na OH )
Will react with glucose produced
Cytology
Eukaryotes
Organelles
Plasma membrane
Nucleus
Cytoplasm = cytosol
Mitochonderia
Ribosomes
Endoplasmic reticulum
Golgi apparatus
Nucleolus
8
Rat liver ( 1 g )
0.25 M sucrose
Homogenization ( Homogenizer ) 1:5
Centrifugation ( Centrifuge )
1. Bench- Top centrifuge ( up to 6000 rpm )
rpm : round per minute
2. High speed centrifuge ( up to 20000 rpm )
Sorvall : American company
3. Ultra centrifuge ( up to 100000 rpm )
Physiological Saline = 0.9% NaCl
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Dehydrogenases enzymes
e.g . isocitrate dehyrogenase
alpha – ketoglutarate dehydrogenase
Oxidoreductases E.C.1
Enzyme Commission
pyruvate dehydogenase complex
Coenzymes
NAD , NADP , FAD , NADH , NADPH , FADH 2
A = KCL
C = A / KL
C = 0.055 * 10 6 µ mol * Cm / 6.22 * 10 3 L * 1Cm
C = 0.055 * 10 6 µ mole * Cm / 6.22 * 10 3 103 ml * 1 Cm
No. of µmoles produced of NADH / min / vol. added of mitochondrial
fraction
U/ml
U / g wet tissue
E 1M1Cm for NADH ( NADPH ) at 340 nm = 6.22 * 10 3 L mol-1 Cm -1
Zero time A = 0.726
1 min.
= 0.772
2 min
= 0826
3 min
= 0.872
4 min
= 0.934
5 min
= 1.00
10
0.046
0.054
0.046
0.062
0.066
∆ A / min = 0.055
Some Lysosmal enzymes
Proteases ( for protein degradation )
Nucleases ( eg, ribonucleases , deoxyribonucleases )
Lipases ( for lipid degradation )
Galactosidases ( α – galactosidases , β- galactosidases )
Phosphatases ( for phosphate removal )
Lysosomal storage diseases ( LSD )
11
Cytosol fraction
18,000 rpm ( High speed centrifuge )
Marker : lactate dehydrogense LDH
Sod. Pyruvate + NADH + H + → lactate + NAD +
CH3 C=O COO- Na + + NADH + H + → CH3 CHOH COO- + NAD +
P→L
L→P
A = KCL
C = A / KL
C = 0.036 * 10 6 µ mol * Cm / 6.22 * 10 3 L * 1Cm
C = 0.036 * 10 6 µ mole * Cm / 6.22 * 10 3 103 ml * 1 Cm
No. of µmoles disappeared of NADH / min / vol. added of cytosolic
fraction
U/ml
1 : 5 ( 1g / 5 ml )
1ml = 0.2 g
U / g wet tissue
E 1M1Cm for NADH ( NADPH ) at 340 nm = 6.22 * 10 3 L mol-1 Cm -1
Zero time A = 0.726
1 min.
= 0.682
2 min
= 0.640
3 min
= 0.60
4 min
= 0.31
5 min
= 0.12
12
0.044
0.042
0.040
0.029
0.019
GOUT ( disorder of uric acid metabolism )
Cofactor
1. Coenzyme
2. Metal ion ( Mg , Zn , Ca …… etc )
3. Prosthetic group
Hydrophilic
Hydrophobic
GluT2
Hydrology
13
Microsomal & lysosomal fraction
Ultra centrifuge ( 75000 rpm )
Marker or indicator
Microsomal fraction
1. Glucose -6- phosphatase ( G6Pase )
G6p → Glucose + Phosphate ( Pi )
Phosphate ( Pi ) +Ammonium molybdate → Phosphomolybdate ( Mo+6 )
+ Reducing agent ( Vit.C : ascorbic acid ) → ( Mo+3 ) Mo2O3
Molybdenum blue
Specificity
Mo : molybdenum
2. MFO : Mixed Function Oxidases ( drug metabolism )
Xenobiotics
Aldehyde oxidase
Xanthine oxidase
14
Lysosomal fraction
Indicator : Acid phosphatase ( ACP)
P- nitrophenyl phosphate → P-nitrophenol + Pi
Yellow in color
Λmax = 400 – 405 nm
Increase in A
Extinction coefficient = 18.7
Prostate cancer ( clinical application )
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