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

1  ما المقصود ؟؟

هل يمكن عزل جميع عضيات الخلية في صورة سليمة ؟؟؟

عزل النواة - التحقق من عزل النواة

عزل الميتوكوندريا - التحقق

عزل الميكروسومات و الليسوسومات - التحقق

عزل السيتوسول – التحقق

تقنية الطرد المركزي - النواع – الخصائص

النزيمات - فكرة عامة - كيفية تقديرها – نشاطية النزيم

الحسابات و النتائج

M – 0.34 M Sucrose solution 0.25

M.W = 342

ml 100

g = 0.25 * 342 * 100 / 1000

g / 100 ml 8.5

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 )

تنقية Purification

Nucleoproteins ( conjugated protein)

Histones

1% E 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

1% E 1Cm for RNA at 260 nm = 250 ( yeast )

Results :

? mg DNA ( or RNA) / g liver dry tissue

أنواع كروماتوجرافيا العمود Column chromatography

( .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

6 Glycolysis

Glucose ______pyruvate

Pyruvate + NADH ______lactate + NAD

LDH : lactate dehydrogenase

RCF : Relative centrifugal force

xg = ??? rpm 45,000

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

7 ( C6H12O6 ( 72 + 12 + 64 = 180

µ mole produced / min / ml E = U / ml U : unit

DNS 3,5

Dinitrosalicylate , yellow – orange in color ( color R for reducing 3,5 ( 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

8 Ribosomes

Endoplasmic reticulum

Golgi apparatus

Nucleolus

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

9 Dehydrogenases enzymes e.g . isocitrate dehyrogenase alpha – ketoglutarate dehydrogenase

Oxidoreductases E.C.1

Enzyme Commission pyruvate dehydogenase complex

10 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

1M 3 -1 -1 E 1Cm for NADH ( NADPH ) at 340 nm = 6.22 * 10 L mol Cm

Zero time A = 0.726

1 min. = 0.772

2 min = 0826

3 min = 0.872

4 min = 0.934

5 min = 1.00

11 0.046

0.054

0.046

0.062

0.066

∆ A / min = 0.055

Some Lysosmal enzymes

12 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 )

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

13 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

1M 3 -1 -1 E 1Cm for NADH ( NADPH ) at 340 nm = 6.22 * 10 L mol Cm

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

0.044

14 0.042

0.040

0.029

0.019

GOUT ( disorder of uric acid metabolism )

Cofactor

1. Coenzyme

2. Metal ion ( Mg , Zn , Ca …… etc )

15 3. Prosthetic group

Hydrophilic

Hydrophobic

GluT2

Hydrology

Microsomal & lysosomal fraction

Ultra centrifuge ( 75000 rpm )

Marker or indicator

Microsomal fraction

1. Glucose -6- phosphatase ( G6Pase )

G6p → Glucose + Phosphate ( Pi )

+6 Phosphate ( Pi ) +Ammonium molybdate → Phosphomolybdate ( Mo )

+3 + Reducing agent ( Vit.C : ascorbic acid ) → ( Mo ) Mo2O3

Molybdenum blue

Specificity

Mo : molybdenum

16 2. MFO : Mixed Function Oxidases ( drug metabolism )

Xenobiotics

Aldehyde oxidase

Xanthine oxidase

Lysosomal fraction

Indicator : Acid phosphatase ( ACP)

17 P- nitrophenyl phosphate → P-nitrophenol + Pi

Yellow in color

Λmax = 400 – 405 nm

Increase in A

Extinction coefficient = 18.7

Prostate cancer ( clinical application )

18