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BIOCHEMISTRY // CELL AND CELL MEMBRANE BIOCHEMISTRY Trans No.: 1 CELL AND CELL MEMBRANE: A
BIOCHEMISTRY // CELL AND CELL MEMBRANE
BIOCHEMISTRY
Trans No.: 1
CELL AND CELL MEMBRANE: A BIOCHEMICAL APPROACH
Lecturer: Dr. Allan Hilario
Date: June 10, 2013
Transcribed by: Balondo, Cahanding, dela Cruz, Dominguez, Farillas, Frias, Montes, Uichanco

BIOCHEMISTRY

A Comparison of Prokaryotes and Eukaryotes

- It is the frontier of science. It is not a dead science. There are still many things unexplored in biochemistry.

- Example: Human genome

- Published in 1986 and only 1-2% of 33,000 enzymes working with billions of base pairs of genomes are used and studied.

- Biochemistry goes hand in hand with molecular biology. Others call it physiological chemistry or the chemistry of life.

- Understanding the biochemical aspect of cell and cell membrane gives us the basis for what comes out as disease or those that do not.

Phylogeny of the Three Distinct Domains of Life

A. Archaebacteria

Extreme halophiles (salt-tolerant)

Methanogens

Extreme thermophiles

B. Eubacteria

Thermatoga

Flavobacteria

Cyanobacteria

Purple Bacteria

Gram-positive bacteria

Green nonsulfur bacteria

C. Eukaryotes

Animals

Plants

Ciliates

Fungi

Flagellates

Microsporidia

 

Prokaryotes

Eukaryotes

Organism

1-10 µm in size i.e. Eubacteria, Archaebacteria

10-100 µm i.e. Fungi, Plants, Animals

Form

Single-celled

Single or multicellular

Organelles,

Missing

Present, complicated, specialized

cytoskeleton,

cell division

 

apparatus

DNA

Small, circular (ALL, with the exception of viruses which can have linear DNA), no introns, plasmids

Large, in nucleus, many introns (evident difference from prokaryotes)

*introns non-coding sequence, not translated

RNA:

Simple, in

Complicated, in nucleus

Synthesis

cytoplasm

and

maturation

Protein:

Simple, coupled

Complicated, in the cytoplasm, and the rough ER

Synthesis

with RNA

and

synthesis

maturation

 
 

Anaerobic (i.e.

Mostly aerobic,

Metabolism

fermentation) or

compartmented

aerobic, very

 

flexible

Endocytosis

No

Yes

and

exocytosis

*enveloped by a thick cell wall

The macromolecule which is the first biochemical basis of life:

RNA (ribonucleic acid) Biohemical bases:

Differentiation between an Animal and Plant Cell

Animal Cell Plant Cell 2. Protein needs to be acted upon by enzyme Nucleolus +
Animal Cell
Plant Cell
2. Protein needs to be acted upon by enzyme
Nucleolus
+
+
Nucleus
+
+
Cell Introduction
Nuclear
Classification of All Organisms according to Energy Source
and Source of Carbon
+
+
membrane
ER (rough,
+
+
smooth)
Golgi
+
+
apparatus
Ribosomes
+
+
Peroxisomes
+
-
Lysosomes
+
-
Cytoskeleton
+
+
Plasma
+
+
membrane
Vacuole
-
+
Cell wall
-
+
Chloroplasts
-, counterpart is the
mitochondria
+
Starch
-
+
granules
Thylakoids
-
+
Plasmodesma
-, counterparts are
gap junctions, etc.
+
Glyoxysome
-, lysosomes as
counterpart
+

Page 1 of 16

1.

Informational capability to produce framework of protein

BIOCHEMISTRY // CELL AND CELL MEMBRANE

Structure of an Animal Cell

Structure of an Animal Cell
// CELL AND CELL MEMBRANE Structure of an Animal Cell Ex. Peroxisome contains catalase (can attach

Ex. Peroxisome contains catalase (can attach a probe with an antibody against catalase, and find it using ELISA)

Prokaryotic cell

against catalase, and find it using ELISA) Prokaryotic cell *Left box (yellow) – percentage of the

*Left box (yellow) percentage of the cell *Right box (blue) quantity in the cell *Number of mitochondrion depends on the differentiation function of the cell

A. Isolation of Cell Organelles

Fractional Centrifugation way to differentiate the compartments of the cell

1. Lyse the tissue

2. Sieve it through a filter membrane to remove the debris like the whole tissue part

3. First to sediment are the nucleus and the cytoskeleton

mitochondria,

4. Second

are

the

organelles

Ex. E. coli

Nucleodis not encapsulated

The bacteria maintains its shape through the cell wall which contains lipopolysaccharide (LPS) which is a membrane bound lipid (Lipid A) attached to polysaccharides which act as endotoxin, responsible for the symptoms of fever and shock in infected animals and humans by gram (-) bacteria. (septicemia)

 

Gram (+)

Gram (-)

Color

Blue-violet

Pink

Stain

Crystal

Violet

Safranin

(Primary)

lysosomes, peroxisomes; plants chloroplasts

5. Third, plasma membrane and ER fragments, small vesicles, microsomal fraction

6. Last, ribosomes, viruses, macromolecules (polymers of carbohydrates, proteins, etc.)

B. Density Gradient Centrifugation

proteins, etc.) B. Density Gradient Centrifugation  A way to differentiate by gradient. When gradient is

A way to differentiate by gradient. When gradient is known of a particular solution, the particular cellular component will hover within that gradient, then you can know its identity by using its marker molecules

then you can know its identity by using its marker molecules  Crowded inside with ribosomes

Crowded inside with ribosomes and different solutes it makes it easier for solute to communicate, anything that comes in the cell is readily attached to any part of the cell targeted No membranes separating the organelles The plasmid is a different set of gene, usually circular which conveys resistance (the material that jumps from cell to cell to prevent destruction by antibiotics)

A. Components of a Bacterial Cell

Water 60-70%

 Water interacts with most substances in the cell making its interaction with water, a
Water interacts with most substances in the cell making
its interaction with water, a classifying distinction for these
substances (Hydrophobic vs. hydrophilic)
The cell is enveloped by cell membrane serving as a
selective physical barrier
The cell is composed of supramolecular structures
capable of self-assembly
B.Inner look
Inner space of cell is crowded with macromolecules 
provide non-specific stearic repulsion  prevents easy
introduction of molecules inside
Macromolecules  occupy 20-40% (protein is dominant)
Smaller molecules  70% of the space
Crowding affects:
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BIOCHEMISTRY // CELL AND CELL MEMBRANE

 

o

Biochemical reaction rates

 

TWO MAJOR PARTS OF THE CELL

o

Protein folding

o

Protein-protein binding

 

1. Cytoplasm

o

Chromosome structure

-

Portion of the cell enclosed by the cell membrane and outside the nucleus

o

Gene expression

o

Signal transduction

-

Where the cellular organelles are found which are membrane-bound or the macromolecules and other ribosomes which are not membrane-bound

*Why crowding takes less time to interact due to less

distance between cells

Proteins becomes super complexes

-

These organelles are not just simply floating but are linked together by the cytoskeleton of the cell

 

o

act as biological machines that perform work when motor protein subunits bind and hydrolyze nucleotides such as ATP

 

-

Cytosol: fluid portion of the cytoplasm; where most of the biochemical processes occur

 

o

ATP universal currency of cells

-

These include glycolysis, gluconeogenesis, fatty acid synthesis, protein synthesis, part of urea cycle and purine metabolism an amino acid synthesis, NADPH production (pentose phosphate pathway; malic enzyme), isoprenoid and sterol synthesis (early stages), fatty acid synthesis

Energy-induced changeproduce conformational and orderly change in shape of the adjacent subunits causing movement (work)

All cells have the inherent ability to interpret and response to signal from its environment and adjacent cells through signal transduction (ability of the cell to respond to stimuli from the environment)

-

But not all cells will do that. There are differentiation of cells where only gluconeogenesis is present and ketogenesis is absent.

 

-

Nucleoplasm: the cytosol in the nucleus

C.

Biochemical Reactions of the Cytoplasm

Pentose phosphate pathway

Cytoskeleton

Gluconeogenesis

Protein biosynthesis

Fatty acid biosynthesis

Glycolysis, etc.

*In prokaryotes, everything happens in the cytoplasm

- a network of protein scaffolding

- maintains the shape of the cell

- serves as tracts for movement of organelles

- These filamentous protein polymers are categorized into 3 types depending on its size with differing functions:

1. Microfilaments (6-8 nm)

2. Intermediate filaments (10 nm)

3. Microtubules (25 nm)

“These characteristics are recurring theme in both prokaryotes as exemplified by a bacterial cell and eukaryotes as exemplified by an animal cell.”

Metabolism is the totality of cellular activities that generates energy for cellular function, (1) conversion of micronutrients, (2) polymerization of various monomeric components of macromolecules and (3) synthesis and degradation of various macromolecules various and specialized cellular functions.

Anabolism is the synthesis of macromolecules from its monomeric units usually for storage.

- The protein scaffolding assembles as polymers from protein subunits.

- What is that recurring theme from the prokaryotes to the eukaryotes? SELF- ASSEMBLY (They have the same characteristics that make them assemble. The non-

covalent factors that make them assemble: London forces, stearric, Van der Waals, hydrogen bond, dipole- dipole, ionic bond.)

Van der Waals, hydrogen bond, dipole- dipole, ionic bond.) - needs reducing equivalents  Catabolism is
Van der Waals, hydrogen bond, dipole- dipole, ionic bond.) - needs reducing equivalents  Catabolism is

- needs reducing equivalents

Catabolism is the degradation of macromolecules to its

monomeric units usually for energy production, entrance to other metabolic pathways or disposal.

- an oxidative process

CELLULAR ORGANIZATION Eukaryotic cell

- Coming from the three germ layers:

1.

Ectoderm producing the external

2.

Mesoderm mesenchymal

3.

Endoderm mostly neural tissue

- There are over 200 cell types in the human body.

tissue - There are over 200 cell types in the human body. 1. Microfilaments - Actin

1.

Microfilaments

-

Actin

most abundant protein in eukaryotic cells

the protein component of microfilaments

occurs in two forms: a monomolecular form (G- actin, globular actin) and polymer (F-actin, filamentous actin)

G-actin is an asymmetrical molecule with a mass of 42 kDa, consisting of two domains.

As the ionic strength increases, G actin aggregates reversibly to form F actin, a helical homopolymer. G actin carries a firmly bound ATP molecule that is slowly hydrolyzed in F actin to form ADP. Actin therefore also has enzyme properties (ATPase activity: It hydrolyzes ATP to ADP and organic phosphate).

Myosin is the globular protein that interacts with actin in myocytes for muscle contraction.

 It is present in all cells not just in the muscles.
 It is present in all cells not just in the muscles.

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BIOCHEMISTRY // CELL AND CELL MEMBRANE

BIOCHEMISTRY // CELL AND CELL MEMBRANE Toxin found in Amanita phalloides mushroom inhibits decay by binding
Toxin found in Amanita phalloides mushroom inhibits decay by binding to the negative polarity.
Toxin found in Amanita
phalloides mushroom inhibits
decay by binding to the
negative polarity.
Mold toxin inhibits polymerization by binding to the positive polarity
Mold toxin inhibits
polymerization by
binding to the positive
polarity

(Refer to the figure above) This is the single monomer of actin and when they polymerize, they become F actin. The negative end which is actually negative in its electric capacity is inhibited by phalloidin, a toxin. The positive end is inhibited by cytochalasin. These two can be used to prevent the formation of actin so the cell will die, especially in cancer cells.

2. Intermediate filaments

- The intermediate filament belongs to five related

protein family with cell type-specificity. These include:

1. Cytokeratins (ectoderm)

2. Desmin (ectoderm)

3. Vimentin (mesoderm)

4. Glial fibrillary acidic protein (GFAP) (endoderm; in the glial cells of the nervous tissue)

5. Neurofilament (endoderm)

- These proteins have a rod-shape structure at the center called super helix configuration.

- The intermediate filament is produced from 8 protofilaments.

Repository and cellular localization of storage replication and expression of genetic information

Contains 99%of cell DNA (1% in the mitochondria)

Structure:

1. Nuclear Envelope- separates the nucleus from the cytoplasm; hastwo separate bilayer membranes (one inside the other)

- The outer membrane is continuous w/ the ER of the cell cytoplasm

- The space between the two membranes is continuous with the space inside the ER

2. Nuclear Pores- controls the movement of substances

between the nucleus and cytoplasm

3. Nucleolus- contains RNA and proteins of the types found in ribosomes

- Has no limiting membrane

4. Nucleoplasm- highly viscous liquid surrounding the chromosomes and nucleoli

5. Chromatin- composed of DNA and other proteins that make up the nucleus

6. Chromosomes- contains most of the cell’s hereditary units (genes) that controls cell structure and its activities

Functions:

- Control center of the cell

- Contains large amounts of DNA

- Transmits genetic information

- Directs protein synthesis in cytoplasm via various RNA forms

- Contains enzymes for DNA repair, replication, transcription, and mRNA production

Pathways that occurs in the nucleus:

1. Replication

a. Nucleus -99%

b. Mitochondria -1%

2. Transcription

3. Translation

4. Biosynthesis of NAD +

2. Transcription 3. Translation 4. Biosynthesis of NAD + 3. Tubulins - The basic components of

3.

Tubulins

- The basic components of the tube-shaped microtubules are α– and β–tubulin (53 and 55 kDa).

- Thirteen protofilaments form a ring-shape and polymerize to form a long tube.

- Important during mitosis.

polymerize to form a long tube. - Important during mitosis. Nucleolus- ONLY part where rRNA is

Nucleolus- ONLY part where rRNA is synthesized Euchromatin- transcribing part; white part in the electron- micrograph Heterochromatin -dense part; dark part

ORGANELLES 1.Mitochondria - Powerhouse of the cell where, most not all, ATP is produced -
ORGANELLES
1.Mitochondria
- Powerhouse of the cell where, most not all, ATP is
produced
- Self-replicating
2. Nucleus
Largest organelle
Page 4 of 16

BIOCHEMISTRY // CELL AND CELL MEMBRANE

BIOCHEMISTRY // CELL AND CELL MEMBRANE *phospholipids produced in SER are immediately incorporated in the bilayer

*phospholipids produced in SER are immediately incorporated in the bilayer of the SER itself for the formation of transport /ER vesicles and replenish the cell membrane bilayer

3. Golgi Apparatus

Responsible for the packing of proteins in vesicles to be exported out of the cell

- Primitive prokaryotic cell engulfed the bacterium (animal- aerobic; plant-cyanobacterium) which resulted to the double-membrane structure called mitochondria (for animal and chloroplast for plants).

- Explains why mitochondria contains circular DNA which is also present in the bacterium

contains circular DNA which is also present in the bacterium Stuctures: 1. Outer membrane – porous

Stuctures:

1. Outer membrane porous

2. Inner membrane selectively porous

3. Cristae-where electron transport chain and oxidative phosphorylation occur; contains enzymes required for ATP synthesis

4. Intermembrane space

5. Matrix

Proteins-major macromolecule content of mitochondria

1. cis face - fusion of areas of Golgi with ER

2. trans face - site for packaging and sorting

Functions:

- Receives protein from rER via coated vesicles to be further processed, packed and released into the cytoplasm as vesicles

- Synthesizes certain carbohydrates not formed in the ER such as hyaluronic acid and chondroitin sulfate

4. Lyososomes

Contains nucleases, proteases, glcosidases, lipases, phosphatases, sulfatases, phospholipases

All macromolecules that gets into the lysosomes (very acidic in nature) are either catabolised for salvage usage or for waste disposal

Structures:

- Originates from the Golgi apparatus

- Acidic at pH 4.5

- Membrane bound vesicles dispersed in the cytoplasm

- Contains digestive enzymes (hydrolase) and bactericidal agents (ex. lysozyme and lysoferrin)

Functions:

Degradative via endocytosis towards:

a. Damaged cell structures

b. Food particles ingested

c. Unwanted materials (ex. bacteria)

- Responsible for tissue regression after long periods of inactivity

- Responsible for autolysis after cell damage

Pathways in the mitochondria:

1. Tricarboxylic Acid Cycle (Kreb’s Cycle)

2. Beta-Oxidation

3. Part of the Urea Cycle

4. Respiratory Chain

5. ATP Synthesis

Transport Systems in the Mitochondria:

1. Citrate-Malate Shuttle

2. Carnitine Shuttle

3. Glyerophosphate huttle

4. Malate shuttle

2. Rough and Smooth Endoplasmic Reticulum

5. Peroxisomes

Degrades peroxides because of the enzyme oxidases present in it

Structures:

- Membrane bound

- Forms by self replication

- Budding off from the sER

- Contains oxidases instead of hydrolases

Functions:

Membrane-bound vesicles containing oxidative enzymes for:

- Detoxifying harmful substances

- Oxidation of fatty acids

A. Rough ER: Granular Endoplasmic Reticulum

Pathways in Peroxisomes:

1. Fatty Acid Oxidation (alpha, beta, omega)

2. Synthesis of certain membrane lipids

3. Purine catabolism

4. Generation and breakdown of NaOH

CELL MEMBRANE

Emerged with most polar charged substances but allows the hydrophobic substances or the non polar substance

A 5 to 10 nanometers in thickness

Appears trilaminar structure when viewed in electron microscope.

- Flattened sacs of ER studded with ribosomes (where protein synthesis occurs) giving its rough appearance

Functions:

- Protein synthesis via ribosomes

- mRNA translational and post-translational modification of proteins

B. Smooth ER: Granular Endoplasmic Reticulum

- Tubular sacs of ER with no ribosomes attached

Functions:

- Site of lipid and cholesterol synthesis 1. Trilaminar - Drug detoxification - Calcium reservoir
- Site of lipid and cholesterol synthesis
1.
Trilaminar
- Drug detoxification
- Calcium reservoir for cell signaling
- Actual picture of the lipid bilayer under the electron
microscope
- Conversion of hydrophobic into water soluble molecules
- Dense part of phosphate group in phospholipid
- Less dense is the tail of the phospholipid
Page 5 of 16

BIOCHEMISTRY // CELL AND CELL MEMBRANE

- The other dense part is the other polar group in the inner layer

2. Lipid Bilayer

Characteristic of the membrane

FLUID MOSAIC MODEL

1972 by Singher and Nicolson

Lipid bilayer wherein the lipid layer is DYNAMIC. There is a lot of movement in the inner and outer leaves.

Proteins and other structures in the bilayer moves. They “float in a sea of lipids.”

Integral membrane proteins are inserted into the lipid bilayer, whereas peripheral proteins arebound to the membrane indirectly by protein-protein interactions. Most integral membraneproteins are transmembrane proteins, with portions exposed on both sides of the lipid bilayer.The extracellular portions of these proteins are usually glycosylated, as are the peripheralmembrane proteins bound to the external face of the membrane.

LIPID CONTENT OF PLASMA MEMBRANE

Major content of the lipid bilayer is PHOSPHOLIPIDS

POLAR: Phosphate group; “head”

NON-POLAR: Fatty acyl chain; “tail”

Arranged in such a way that the outer polar group is in contact with the outer matrix and the inner polar group is in contact with the cytoplasm.

Middle contains the non-polar acyl chains of both tails.

They are attached by non-covalent bonds due to their stearic attraction.

In general, most commonly found are: Phospholipids, sphingolipids and cholesterol.

*Is the lipid content of plasma membrane constant in all cell types? No, they vary.

II. PROTEIN MOVEMENT

in all cell types? No, they vary. II. PROTEIN MOVEMENT 1. Cells from two different species

1. Cells from two different species (human and mouse, blue and pink respectively) are tagged with fluorescent antibodies that react with membrane proteins.

2. These cells are induced to fuse by exposure to Sendai virus. The proteins from the two parent cells are localized to two separate halves of the hybrid cell initially.

3. Within a few minutes, they begin to intermix.

4. After around 40 minutes, they are now randomly distributed in the plasma membrane.

PROTEINS IN THE CELL MEMBRANE

in the plasma membrane. PROTEINS IN THE CELL MEMBRANE GLYCOPROTEINS: outer leaflet Oligosaccharides: outer leaflet

GLYCOPROTEINS: outer leaflet Oligosaccharides: outer leaflet

CELL MEMBRANE MOTION STUDIES

I. LIPID MOVEMENT

Important for signal transduction and recognition

Antigen-antibody binding

transduction and recognition  Antigen-antibody binding FUNCTIONS OF THE CELL MEMBRANE  The bounding function of

FUNCTIONS OF THE CELL MEMBRANE

 Antigen-antibody binding FUNCTIONS OF THE CELL MEMBRANE  The bounding function of the membrane gives

The bounding function of the membrane gives the cell its shape and separates itself from other cells and external environment.

When there is actual anchorage of any protein in the cell, there is less movement.

CELL MEMBRANE LIPID COMPOSITION

1. The cell was reacted with fluorescent probe to label the lipids of the cell
1. The cell was reacted with fluorescent probe to label the
lipids of the cell membrane (first step).
2. Second step shows the fluorescent labeled cell
membrane and its appearance in the fluorescence
microscope.
3. In the third step, an intense laser beam bleaches a small
area of the cell membrane and its appearance
microscopically.
4. In time, the unbleached phospholipids diffuse into the
bleached area (therefore, movement).
Page 6 of 16

BIOCHEMISTRY // CELL AND CELL MEMBRANE

It

be

phosphatidylserine.

can

generalized

that

the

inner

leaflet

contains

2. Lipid bilayer  Basic structural element of the cell membrane. 3. Vesicle/Liposome
2.
Lipid bilayer
Basic structural element of the cell membrane.
3.
Vesicle/Liposome
element of the cell membrane. 3. Vesicle/Liposome  Tricylglycerol or neutral fat is not present in
element of the cell membrane. 3. Vesicle/Liposome  Tricylglycerol or neutral fat is not present in

Tricylglycerol or neutral fat is not present in cell membranes with only a few exceptions.

Cholesterol is exclusively found in animal cell membranes. When present in plants, they are called steroids.

CELL MEMBRANE COMPOSITION IN DIFFERENT CELLS

steroids. CELL MEMBRANE COMPOSITION IN DIFFERENT CELLS TYPES  NERVE CELL PLASMA MEMBRANE o

TYPES

NERVE CELL PLASMA MEMBRANE

o

Sphingomyelin is high because it serves as an insulator (i.e. myelin sheath)

MITOCHONDRIA

 

o

high in proteins because these proteins are enzymes responsible for oxidative phosphorylation and the electron transport chain

 

CARDIOLIPIN

 

only specialized lipid that is antigenic; present in the mitochondria

proof of endosymbiosis

 

SELF-ASSEMBLY OF PHOSPHOLIPIDS

Phospholipids when placed in an aqueous solution can assemble itself in different ways.

1.

Micelle

 
itself in different ways. 1. Micelle    Two layers of lipid bilayer engulfing an aqueous
 Two layers of lipid bilayer engulfing an aqueous environment.  Presence of an aqueous
Two layers of lipid bilayer engulfing an aqueous
environment.
Presence of an aqueous cavity inside the inner membrane.
CELL MEMBRANE FLUIDITY
The most important characteristic of the fluid mosaic model
is its fluidity.
Fluidity refers to the viscosity of the lipid bilayer or the
degree by which the bilayer resists movement.
Factors Affecting Membrane Fluidity
1. Temperature
- Hydration of polar heads and the tails aggregate in the
center
Higher temp, higher fluidity
- No cavity inside
2. Unsaturated Fatty Acid Content
- Individual units are wedge-shaped (cross section of
head greater than that of side chain)
More unsaturated fat, more fluid
Page 7 of 16

BIOCHEMISTRY // CELL AND CELL MEMBRANE

Presence

of

double

bonds,

therefore

kinks

in

Cell Membrane Self-Sealing

hydrophobic tail.

kinks

stacking.

The

can’t provide an ordered structure by

This is for the maintenance of cell membrane integrity. Two types:

a. Lateral diffusion of lipid contents for small disruptions

b. Energy requiring process through Ca2+ for larger tear due to mechanical stress

3. Cholesterol (paradoxical)

Aliphatic: polar and nonpolar

Paradoxical way by which it provides fluidity.

Cell Membrane Fluidity

Viscosity of the lipid bilayer or the degree of resistance of membrane components to move.

Factors:

1. Temperature: higher T, more fluid

2. Content of unsaturated FA: higher content, more fluid

3. Content of cholesterol: when cholesterol interacts with unsaturated/short-chain fattyacyl PL results to less fluidity; when cholesterol interacts with spingolipids and long-chain fattyacyl PL tends to make it more fluid.

Cell Membrane Lipid Movement

1. Rotation

make it more fluid. Cell Membrane Lipid Movement 1. Rotation 2. Lateral diffusion - very fast

2. Lateral diffusion - very fast

Lipid Movement 1. Rotation 2. Lateral diffusion - very fast 3. Flip flop - very slow

3. Flip flop - very slow that enzymes are needed

- very fast 3. Flip flop - very slow that enzymes are needed Cell Membrane Selective

Cell Membrane Selective Permeability

The hydrophobic hydrocarbon chains in lipid bilayer provide an impervious barrier for ionic and polar substances.

Specific proteins - regulate the passage of ionic and polar substances in and out of the cell so that polar groups cannot pass through easily the cell membrane but hydrophobic groups can easily diffuse:

Water - aquaporins

Polar substances - shed hydration shell

Large molecules and ions - transport system such as protein membrane

and ions - transport system such as protein membrane Cell Membrane Asymmetry  The inner and

Cell Membrane Asymmetry

The inner and outer leaflets of the lipid bilayer differ in composition

Components of phospholipid that needs enzyme:

1. Flippase

- P-type ATPase

- Allows one polar head group to go the other side

- Requires energy

2. Floppase

- ABC transporters

- Inner leaflet to the other side

- Requires energy

3. Scramblase

- Moves either way

- ABC transporters - Inner leaflet to the other side - Requires energy 3. Scramblase -
- ABC transporters - Inner leaflet to the other side - Requires energy 3. Scramblase -

Page 8 of 16

BIOCHEMISTRY // CELL AND CELL MEMBRANE

Cell Membrane Proteins

Cell Membrane Microdomains

These are for transfer of solute. The latter cannot enter the lipid bilayer because it is polar.

Examples:

1. Lipid Raft

lipid bilayer because it is polar. Examples: 1. Lipid Raft Classification: 1. Integral proteins - Embedded
lipid bilayer because it is polar. Examples: 1. Lipid Raft Classification: 1. Integral proteins - Embedded

Classification:

1. Integral proteins

- Embedded in and/or pass through the lipid bilayer

- Extracted only through disruption of the cell membrane with organic solvents or detergents

- Amino group in cytoplasm or extracellular side

- Traverse the membrane in a certain manner wherein the hydrophobic part of the segment of the polypeptide traverse in certain type of manner the cell membrane making a ionic channel wherein solutes can pass through

- Allow hydrophobic interaction with lipid bilayer

- Most have alpha-helical structures with 15 to 20 aa

- Have bulky side chains: alanine, leucine, isoleucine, phenylalanine, and valine

- May have several helices that pass through the membrane in serpentine manner

- Thickened portion of the lipid bilayer comprising about 50% and rich in cholesterol and sphingolipids

- Rich in sphingolipids and cholesterol where the GPI- anchoring protein can be seen

- Important in anchorage and cell-cell interaction and signaling

2. Caveola

anchorage and cell-cell interaction and signaling 2. Caveola 2. Transmembrane proteins (but not integral proteins) -

2. Transmembrane proteins (but not integral proteins)

- Beta-pleated sheet is the secondary structure:

a. FepA-ion transport for bacteria

b. OmpLA-phospholipase in E. coli

c. Maltoporin- transport in E.coli

d. In human: porins in outer membrane of mitochodria

3. Peripheral proteins

- Bound to the membrane through non-covalent interaction with integral proteins

- Covalently bound to several mechanism: myristic, palmitic, prenyl group, glycosylphosphatidylinositol anchors (GPI-anchors) in lipid rafts

- Can also be found in extracellular

Hydropathy plot - a graph determining the sequence of amino acid in polypeptide will have a certain hydrophobicity

- - Endocytosis and Exocytosis  For bigger substances to transport
-
-
Endocytosis and Exocytosis
 For bigger substances to transport

Microdomains - 50% of proteins in cell membrane

Curved lipid raft, due to the action of caveolin, a protein which dimerizes with other caveolin through its fatty acyl and cholesterol moities, producing a strain and causing the cell membrane to invaginate.

Important in various cellular process involving membrane breakage and sealing, and signal transduction.

membrane breakage and sealing, and signal transduction. a. Endocytosis - the entrance of substances, smaller

a. Endocytosis - the entrance of substances, smaller macromolecules, and other debris through a cell membrane mediated engulfment without leakage of

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BIOCHEMISTRY // CELL AND CELL MEMBRANE

cell contents or without integrity

the loss of cellular membrane

contents or without integrity the loss of cellular membrane b. Exocytosis - the release of substances,

b. Exocytosis - the release of substances, smaller macromolecules,and other substances for transport to othe issues and cells through a celmembrane mediated release without leakage of cell contents or without the loss of cellular membrane integrity

Cell Moembrane Transport

Transfer of solutes and information across membranes

1. Diffusion

2. Simple diffusion (passive and facilitated)

3. Cross-membrane movement of large molecules

4. Endocytosis

5. Exocytosis • Signal transmission across membrane

Cell surface receptors

1)

2) Signal internalization (coupled with endocytosis like

Signal transduction (glucagon cAMP)

LDL receptos, Insulin

and Glut transporters)

Movement to intracellular receptors (steroid hormones, thyroid hormones, retinoids, Vit. D hydrophobic in nature so they do not need transport proteins; easily pass through the membrane)

Creation of Electrochemical Gradient

Diffusion

Simple diffusion without membrane protein - movement of solutes down its electrochemical gradient due random thermal movement or simply because the solute is permeable through the lipid bilayer because it is small enough and/or hydrophobic

Facilitated diffusion - movement of solutes down its electrochemical gradient through either transporters or ion channels (membrane proteins)

How do transporters allow the passage of hydrophobic solute?

- For proteins to transverse the lipid bilayer, they have to interact with the hydrophobic core within the chain of the sequence of amino acids protrudes out the hydrophilic part, e.g. the hydrogen and nitrogen, making the core ionic in nature so hydrophilic solute can pass through

- By lowering the energy of activation of the solute (ex. hydrated Na + ion needs much energy to pass through the membrane but transporters lower the free energy change)

Passive and Active Transport

lower the free energy change) Passive and Active Transport Free diffusion – H2, CO2, urea, O2

Free diffusion H2, CO2, urea, O2 Facilitated diffusion downhill but does not need energy

Depends on the chemical gradient (or the difference of solute concentration or its ratio C2/C1) and the transmembrane electrical gradient, Vm in millivolts. Solutes follow the 2nd law of thermodynamic where it tends to assume spontaneously the greatest randomness and the lowest energy

Comparison between Transporters and Ion channels

1. Transporter- alternating gates

- Binds molecule and ion with high specificity

- Undergoes conformational change to let the transported ion enter the other side of the membrane

- Saturable/ acts like an enzyme - Involves active and passive transport (facilitated diffusion) 2.
-
Saturable/ acts like an enzyme
-
Involves active and passive transport (facilitated
diffusion)
2.
Ion Channels – single gate
-
Show some specificity for the solute but does not act like
an enzyme and only forms pores which open and close
with less conformational change
-
Non-saturable
-
Involve mostly facilitated diffusion
Uniport, Symort, Antiport
A.
Glucose - through passive transport and a uniport which is
used in transporting glucose in the cell through the influence
of insulin
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B. Glucose - may also be transported together with sodium into cell as a symport type of facilitated transport

C. Chloride-bicarbonate exchanger of the RBC membrane - allows the transport of bicarbonate without changing the membrane potential in opposite direction with chloride ions and increases the carbon dioxide carrying capacity of blood

and increases the carbon dioxide carrying capacity of blood Aquaporins  Water channels that only allow

Aquaporins

Water channels that only allow the passage of water molecules in the membranes of RBC and cells of the collecting ductules of the kidney

Mutation in some of these channel proteins may cause Nephrogenic Diabetes Insipidus [a disorder in which a defect in the small tubes (tubules) in the kidneys causes a person to pass a large amount of urine].

Comparison between Ionotropic Channels and Ionophores

Ionotropic Channels

Ionophores

Receptors that acts as channels and do not need a secondary messenger

Non-receptor compounds that produce channels or pores in the cell. If it produces pores or channels in bacteria they serve as anti-bacterial. While if they cause production of pores or channels on cell membranes of human cells, they cause injury or disease.

a. Acetylcholine- Na+ and

Valinomycin-K+ channels Monensin-Na+ channels

Ca2+

b. Serotonin and

Gramicidin-folding creates hollow channels

Glutamate-K+, Na+,

Ca2+

 

c. Glycine-Cl - specific channels

a. Dendrotoxin (mamba snake)- K +

Diptheria toxin and activated complement- create large cellular

b. Tetrodotoxin (puffer fish)- Na+

pores causing lysis Alpha-hemolysis (Strep.), leak out

c. Cobrotoxin and alpha

ATP

conotoxin-

d. Acetylcholine receptor

ion channel

TERMS

Secondary Active transport- occurs when endergonic (uphill) transport of one solute is coupled to an exergonic (downhill) flow of a different solute that was originally pumped uphill a primary active transport

that was originally pumped uphill a primary active transport Diagram transport mechanisms. (directly) above summarizes
that was originally pumped uphill a primary active transport Diagram transport mechanisms. (directly) above summarizes
that was originally pumped uphill a primary active transport Diagram transport mechanisms. (directly) above summarizes

Diagram

transport mechanisms.

(directly)

above

summarizes

cell

membrane

Active transport - the diffusion of molecules and ions against its electrochemical gradient with the use of energy which is mostly supplied by ATP; its protein transporter has an ATPase activity hydrolyzing ATP to ADP producing the needed energy.

Primary Active Transport - the solute accumulation coupled directly to an exergonic chemical reaction such as breakdown of ATP

to an exergonic chemical reaction such as breakdown of ATP Venom of animals like snakes targets

Venom of animals like snakes targets the ion channels instead of proteins of metabolism. If it wanted to take down another organism, it would inhibit the other’s glycolytic pathway. Ion channels and the metabolic pathway are both composed of proteins (enzymes n the metabolic pathway). *In science the straight forward line going to

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Importance of Ion Channels

BIOCHEMISTRY // CELL AND CELL MEMBRANE

the answer is the easiest and most correct one*. In the evolutionary sense it is always through the conservation that our species (or other species) perpetuate. Animals with poisons want to make their venom more effective by targeting ion channels since even when targeting those alone the venom will already affect numerous parts of the body. In the case of action potentials, when these are disturbed/interrupted an animal/human may be paralyzed right away. Paralysis due to targeting proteins of the glycolytic pathway may take more time compared to the immediate paralysis by disruption of ion channels.

Cell Membrane Transport

Some types of ATP-deriven Active Transporters

Type

Example

Cellular Location

Ca 2+ ATPase

P-Type

Sarcoplasmic

Reticulum

N + -K + ATPase

Plasma or cell membrane

F-Type

mt ATPase

synthase of

oxidative

phosphorylation

Mitochondria

V-Type

The ATPase that pumps protons into lysosomes and synaptic vesicles

Lysosomal and

synaptic vesicular

membranes

ABC

Transported

CFTR protein

Plasma or cell membrane

MDR I protein

Plasma or cell membrane

Some of the types of ATPase (from Harper) are listed on the table above. The most important to remember is your sarcoplasmic reticulum, a specialized form of endoplasmic reticulum in the myocyte. This facilitates occurrence of muscle contraction.

Ion Channels and Electrical Signals in Excitable Cells

The membranes of nerve cells contain well-studied ion channels that are responsible for the generation of action potentials. The activity of these channels is controlled by neurotransmitters; hence, channel activity can be regulated (Murray, R.K., Bender, K.M., Kennelly, P.J., Rodwell, V.W., Weil, P.A., 2012).

Ion channels are open transiently and thus are “gated”. Gates can be controlled by opening or closing. In ligand-gated channels, a specifc molecule binds to a receptor and opens the channel. Voltage-gated channels open (or close) in response to changes in membrane potential. Mechanically-gated channels respond to mechanical stimuli like pressure or touch (Murray, R.K., Bender, K.M., Kennelly, P.J., Rodwell, V.W., Weil, P.A., 2012).

Characteristics of ion channels are listed in Tables 40-4 and 40-5 of Harper.

ion channels are listed in Tables 40-4 and 40-5 of Harper. An action potential is one

An action potential is one that causes the repolarization of the cell membrane of the axon which may lead to activation of post- synaptic vesicles of the post-synaptic neurons, or the contraction of muscles which is brought about by the ionic composition within the cell and the external environment. In the following Table 40-1, we find that the extracellular fluid is very high in sodium (140 mmol/L) however the intracellular fluid only has 10 mmol/L of Na + . The potassium (K + ) on the other hand is very high in the intracellular fluid (140 mmol/L) while very low (only 4 mmol/L) in extracellular fluid. There is a discrepancy between sodium and potassium levels. Mataas ang sodium sa labas, mababa sa loob; mataas naman ang potassium sa loob, mababa sa baba. Similarly so, Calcium is like Sodium and Chloride because they are depolarizing ions. Remember this, the sodium, calcium, and chloride ions are depolarizing.

the sodium, calcium, and chloride ions are depolarizing. Dr. Hilario introduced the ion-gated channels just to
the sodium, calcium, and chloride ions are depolarizing. Dr. Hilario introduced the ion-gated channels just to

Dr. Hilario introduced the ion-gated channels just to give us the principle behind them (but this would be discussed in more detail in physiology). And asked the question: What kind of transport is this (Sodium-potassium ATPase)? Anti-core, primary active transport! For the action of this transporter, for every three potassium ions out, enters two sodium ions (see schematic diagram that follows).

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BIOCHEMISTRY // CELL AND CELL MEMBRANE

membrane
membrane

There is an imbalance: there is negativity inside the cytoplasm and positivity outside it. This is the membrane potential which is about - 50 to - 70 mV. So any perturbation in the cell membrane like pinching or any injury or stimulation will cause the leakage of sodium through the sodium channels where it goes inside causing depolarization of the membrane (Ano yung tatlong ion na nag-de- depolarize? Those are sodium, chloride, and calcium ions). So this time sodium depolarizes the membrane. It goes through the membrane until it reaches the neuromuscular junction. It will then depolarize the vesicle that releases the Acetylcholine (ACh) which will then attach to the receptor in the post-synaptic axon. Depolarization of sodium will continue until it reaches the neuromuscular junction and release the calcium through the T- tubule so that it propagates the action potential.

the T- tubule so that it propagates the action potential. Problem with cystic fibrosis: secretions are

Problem with cystic fibrosis: secretions are thick because there’s a mutation in the gene encoding the CFTR protein which is a chloride transporter. When chloride passes through the ion channel what comes with it? Sodium does. Water follows sodium. Water hydrates the lining of the respiratory membrane. Without water, the lining becomes thick; bacteria tend to be trapped in it. Most of cystic fibrosis patients die of pneumonia.

DISEASES INVOLVING ION CHANNEL DEFECTS

Table 11-7 below lifted from Dr. Hilario’s lecture notes. Note emphasized items.

The neuron starts the action potential. It’s the same membrane in the diagrammatic scheme (see second to the last page). It goes to the neuromuscular junction and the muscle contracts causing the movement. If you look at the very small part of the axon, it would reach the neuromuscular junction and the neuron will act on the skeletal muscle.

You can see the release of the neurotransmitter, ACh in the diagram, so that it would act on the post-synaptic vesicles. Post- synaptic vesicles will then continue the action potential wherein sodium depolarizes the cell membrane. The acetylcholine binds to the receptor of the motor end plate of the neuromuscular junction. This depolarizes the cell membrane of the muscle fiber and going to the tubules releasing the calcium from the sarcoplasmic reticulum. The T-tubules are invaginations of the cell membrane. The same sodium channel acts on the membrane. It then releases the calcium. When there’s calcium it will stimulate the troponin until it works its way through actin-myosin binding so that there is movement. The sarcomere shortens thus causing the contraction. It moves along until it provides you the movement. Sodium is the one that leads the action potential. Chloride and calcium also depolarizes while potassium hyperpolarizes.

This diagram presents Ach as a receptor in a ligand-gated channel (two images in the last page). The others formerly presented are ion-gated channels.

DISEASES INVOLVING ABNORMALITIES OF MEMBRANES

Table 40-7 from Harper’s lists diseases involving abnormalities in Cell Membranes. Those enclosed in red rectangles are the ones emphasized by Dr. Hilario.

in Cell Membranes. Those enclosed in red rectangles are the ones emphasized by Dr. Hilario. Page

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BIOCHEMISTRY // CELL AND CELL MEMBRANE

For the ion channel defects, we have the sodium-gated channel for hyperkalemic periodic paralysis (Dr. Hilario doesn’t want us to know the clinical part here. Just associate it with ion channels so that you can at least know the relevance of studying these diseases in relation to the ion channels). Ion channels defects of skeletal muscle responsible for the clinical entity: hyperkalemic periodic paralysis.

ARTIFICIAL MEMBRANES AND THEIR IMPORTANCE

paralysis. ARTIFICIAL MEMBRANES AND THEIR IMPORTANCE When you look at the solution of aliphatic hydrocarbons in

When you look at the solution of aliphatic hydrocarbons in solution with aqueous membranes you will find the electron micrograph like this (as in image above). Each layer is a bilipid layer. When you subject this to ultrasound frequency but mostly sonication, it creates a vesicle-like structure where there is an aqueous cavity inside. But still it is bounded by the lipid bilayer.

IMPORTANCE OF ARTIFICIAL MEMBRANES

1. Drug delivery where liposome, being hydrophobic in nature, can easily enter the normal cell, cancer cell, and bacterial cell

So anything that can be dissolved in aqueous solution (that you can put inside the cavity) can be transported through the membrane so easily. Anti-cancer drugs, which are usually hard to transport through the membrane because of their ionic character, can be easily transported by liposome. The importance of liposome is mainly (at the moment) for drug delivery. Liposome being hydrophobic in nature can easily enter the cell, i.e. cancer cells and bacterial cells. There are liposomal preparations of doxorubicin which is a neomycin that is anthracite-based chemotherapeutic agent for most solid tumors, and cancer. There is also liposomal preparation of ticarcillin and tobramycin which are used on most resistant bacteria that resist usual preparations of those antibiotics. With the presence of liposomal carriers they can easily penetrate the membrane and gain access to bacteria in order to facilitate bacterial destruction.

2. Gene therapy liposome can easily penetrate the nucleus that gene insert may be easily incorporated.

3. The Phospholipid (PL) may be attached to an antibody where drug delivery may be target-oriented.

Anything may be attached to phospholipids which are ionic parts of the cell membrane. You can attach protein which attach to antibodies so you can target whatever you want to target (most especially cancer cells).

4. Studies and researches on the activity and property of various lipid and protein components of the cell membrane will soon be made possible.

References:

Dr. Allan Hilario Lecture and Notes

Murray, R.K., Bender,K.M., Kennelly, P.J., Rodwell, V.W., Weil, P.A. (2012). Harper’s Illustrated Biochemistry (29 th ed.). North America: McGraw Hill Companie, Inc.

Images:

Dr. Allan Hilario Lecture and Notes

http://www.zoology.ubc.ca/~gardner/synapses%20-

%20presynaptic.htm

http://www.cnsforum.com/imagebank/item/rcpt_sys_nic_ag1/de

fault.aspx

Gene therapy has failed before since most vectors used are HIV viruses (retroviruses). E ‘diba natatakot ang tao, pag retrovirus HIV yun. So how could you prevent infection with HIV if the vectors for gene therapy are HIV viruses? So the first experiment for gene therapy of ADA (adenosine deamylase) for severe combined immune deficiency disease was cancelled.

In the future, liposomal insertion (gene insert) into the chromosome through the nucleoplasm through the
In the future, liposomal insertion (gene insert) into the
chromosome through the nucleoplasm through the nuclear
membrane may be easily achieved through liposomal carriers.

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BIOCHEMISTRY // CELL AND CELL MEMBRANE

Metabolic Definition Function Cellular Location Terms Glycolysis Breakdown of glucose and other hexoses to
Metabolic
Definition
Function
Cellular Location
Terms
Glycolysis
Breakdown of
glucose and other
hexoses to pyruvate
or lactate
Production of ATP for
energy need of the ell and
for lipogenesis
Cytoplasm of all cells
Gluconeogenesis
Synthesis of glucose
from glucogenic
precursor
Use of glucose to undergo
glycolysis for energy need in
case of scarce food source
Cytoplasm of hepatocytes and
renal cells; and in special
conditions intestinal cells
Hexose
Monophosphate
Breakdown of
glucose and other
hexoses for the
production of
reducing equivalent
and the sugar ribose
Cytoplasm of all cells
Shunt
Provide the reducing
equivalents for most
synthetic pathways and the
sugar backbone (ribose) for
DNA and RNA synthesis
Breakdown of
Glycogenolysis
glycogen into
glucose
To provide glucose readily
from its storage form
(glycogen) for energy need
Cytoplasm of hepatocytes and
muscle cells
Synthesis of
Glycogenesis
glycogen from
glucose
To store excess glucose
from the diet in glycogen
form
Cytoplasm of hepatocytes and
muscle cells
Tricarboxylic
Acid Cycle or
TCA or Kreb’s
Cycle
Central metabolic
pathway of all cells
To burn the acetyl-CoA
made from fat, glucose, or
CHON to make ATP in
cooperation with oxidative
phosphorylation
Mitochondria of all cells (partly
cytoplasm)
Oxidative
phosphorylation
and the electron
transport chain
Pathway for
producing the ATPs
from all electron
contributing
pathways for energy
needs
To synthesize ATPs from
electrons from NADH and
FADH
Mitochondria of all cells
Lipolysis (Beta-
Degradation of fatty
acids for energy
need
Mitochondria of all cells
Oxidation)
To break down fatty acids
from acetyl-CoA
Lipogenesis
Synthesis of lipids
To synthesize fatty acids
from acetyl-CoA
Fat cells and Part of it in the
cytoplasm
Alpha and Beta-
Oxidation
Same as Beta-
Oxidation
To breakdown FA with more
than 18 carbons
Peroxisomes
Omega-oxidation
Same as Beta-
oxidation
To breakdown medium and
long-chain fatty acids
Peroxisomes
Replication
Synthesis of DNA
To provide duplicate copies
of the DNA for cell division
Nucleus
Transcription
Synthesis of RNA
To serve as a template of
nformation transfer from the
DNA of the genes and for
protein synthesis and the
other types of RNA
Nucleus
Translation
Synthesis of proteins
To synthesize protein gene
expression
(rER) cytoplasm
Post-translational
modification
Modification of
protein molecules
after its synthesis
To provide the protein
molecules its mature
function
Multiple
Protein Sorting
Packaging of protein
either for temporary
storage or transport
To direct the release of
protein to its target of action
Golgi apparatus
Ketogenesis
Excess acetyl-CoA is
converted to ketone
bodies
To provide fuel source
especially the brain during
prolonged starvation
Matrix of mitochondria of
hepatocytes (in cytoplasm);
and in special conditions renal
cells
Urea Cycle
Cyclical pathway that
produces urea from
ammonia,CO 2 and
aspartate
To dispose approximately
90% of surplus nitrogen in
the presence of excess
amino acids as in excess
intake or starvation
Hepatocytes

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BIOCHEMISTRY // CELL AND CELL MEMBRANE

Metabolic

Terms

Definition

Function

Cell Location

Amino Acid

Synthesis

The synthesis of amino acids

To provide the amino acids needed for protein synthesis

rER (Cytoplasm)

Paanong RBC? Once nucleated precursors of RBC are already removed of cytoplasm, the RBC is already complete, it’s just waiting itself to die after 120 days. Only function is to transport haemoglobin.

Amino Acid

Catabolism

Degradation of

amino acids

To provide intermediates for energy needs and as dependent compounds

Cytoplasm of all cells

Volatage-gated Channel

compounds Cytoplasm of all cells Volatage-gated Channel Ligand-gated Channel Ligand-gated Channel *Illustration

Ligand-gated Channel

Ligand-gated Channel

*Illustration lifted from Dr. Hilario’s Lecture Notes*
*Illustration lifted from Dr. Hilario’s Lecture Notes*
Ligand-gated Channel Ligand-gated Channel *Illustration lifted from Dr. Hilario’s Lecture Notes* Page 16 of 16

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