Erythrocyte

Metabolism and
Membrane Structure
and Function
PRESENTED BY:
3A- KRYSTEL MAE ALIA
3B- MARITONNIE MYRIELLE ALTAR
TOPIC OUTLINE

Energy Production—Anaerobic Glycolysis

Glycolysis Diversion Pathways (Shunts)
• Hexose Monophosphate Pathway
• Methemoglobin Reductase Pathway
• Rapoport-Luebering Pathway
RBC Membrane
• RBC Deformability
• RBC Membrane Lipids
• RBC Membrane Proteins
Osmotic Balance and Permeability
• There are approximately 5 million erythrocytes (red blood cells,RBCs)
per microliter of circulating blood making the RBC the primary cell in
the blood. The RBC lacks a nucleus, has a biconcave shape, and has an
average volume of 90 fL.
• The cytoplasm of the RBC contains abundant hemoglobin, which
transports O2 from the lungs to the tissues.
• Hemoglobin has four globin chain, and each chain contains a heme
molecule with an iron in the ferrous state.
• The RBC also transports CO2 and bicarbonate (HCO3) from the tissues
back to the lungs.
• RBCs are produced through normoblastic proliferation and mature in
the bone mmarrow.
• The RBC has a circulating life span to 120 days, whereupon it is
disassembled into its reusable components: globin chains and iron
from hemoglobin, and phospholipids and proteins from the cell
membrane.
ENERGY PRODUCTION—ANAEROBIC GLYCOLYSIS

• Lacking mitochondria, the RBC relies on anaerobic glycolysis for its

energy.
• Hematologists have identified hereditary deficiencies of nearly every
glycolytic enzyme, and their common result is shortened RBC survival,
known collectively as hereditary nonspherocytic hemolytic anemia.
• Anaerobic glycolysis, or EMP (Embden-Meyerhof pathway) requires
glucose to generate ATP, a high-energy phosphate source. RBCs lack
internal energy stores and rely on plasma glucose to enter the cell to
generate ATP.
• Glucose enters the RBC through facilitated diffusion via the
transmembrane protein Glut-1.
• Glucose is then catabolized to pyruvate (pyruvic acid) in the EMP,
generating four molecules of ATP per molecule of glucose, for a net gain of
two molecules of ATP.
Glycolysis is organized into three phases

The first phase of glycolysis employs glucose

phosphorylation, isomerization, and
diphosphorylation to yield fructose 1,6-
bisphosphate (F1,6-BP). Intermediate stages
employ the enzymes hexokinase, glucose-6-
phosphate isomerase, and 6-
phosphofructokinase. The initial hexokinase
and 6-phosphofructokinase steps consume a
total of two ATP molecules and limit the rate of
glycolysis. Fructose-bisphosphate
aldolase then cleaves F1,6-BP to produce
glyceraldehyde3-phosphate
The second phase of glucose catabolism
converts G3P to 3-phosphoglycerate (3-PG).
The substrates,enzymes, and products for this
phase of glycolytic metabolism. In the first
step, G3P is oxidized to 1,3
bisphosphoglycerate (1,3-BPG) through the
action of glyceraldehyde-3-phosphate
dehydrogenase (G3PD) with the reduction of
NAD to NADH. 1,3-BPG is dephosphorylated by
phosphoglycerate kinase , which generates two
ATP molecules and 3-PG.
The third phase of glycolysis converts 3-PG to
pyruvate and generates ATP. Substrates, enzymes,
and products are listed in Table 6.3. The 3-PG is
isomerized by phosphoglycerate mutase to 2-
phosphoglycerate (2-PG). Enolase (phosphopyruvate
hydratase) then converts 2-PG to
phosphoenolpyruvate (PEP). Pyruvate
kinase (PK) splits off the phosphates, forming two
ATP molecules and pyruvate. PK activity is
allosterically modulated by increased concentrations
of F1,6-BP, which enhances the affinity of PK for PEP.
Thus when the F1,6-BP is plentiful, increased activity
of PK favors pyruvate production. Pyruvate may
diffuse from the
erythrocyte or may become a substrate for lactate
dehydrogenase (LD or LDH) with regeneration of the
oxidized form of nicotinamide adenine dinucleotide
(NAD1). The ratio of NAD1 to the reduced form (NADH)
modulates the activity of LD.
 GLYCOLYSIS DIVERSION PATHWAYS (SHUNTS)

• Three alternate pathways, called diversions or shunts,

branch from the glycolytic pathway.

• The three diversions are the hexose monophosphate

pathway (HMP), the methemoglobin reductase pathway,
and the Rapoport-Luebering pathway.
 Hexose Monophosphate Pathway

• Oxidative glycolysis occurs through a

diversion of glucose catabolism into the
HMP, also known as the pentose
phosphate shunt.

The HMP shunt extends the functional

life span of the RBC by maintaining
membrane proteins and lipids,
enzymes, and hemoglobin iron in the
functional, reduced, ferrous state.
• NADPH is then available to reduce oxidized
glutathione (GSSG) to reduced glutathione (GSH) in the
presence of glutathione reductase.
• Glutathione is a cysteine containing tripeptide, and the
designation GSH highlights the sulfur in the cysteine
moiety. Reduced glutathione becomes oxidized as it
reduces peroxide to water and oxygen via glutathione
peroxidase.

• G6PD provides the only means of generating NADPH

for glutathione reduction, and in its absence
erythrocytes are particularly vulnerable to oxidative
damage.
• G6PD deficiency, the most common inherited RBC
enzyme deficiency worldwide, the ability to detoxify is
hampered, resulting in hereditary nonspherocytic
anemia.
 Methemoglobin Reductase Pathway
■ Heme iron is constantly exposed to oxygen and
peroxide. Peroxide oxidizes heme iron from the
ferrous (2+) to the ferric (3+) state.
■ When the iron state is ferric, the affected
hemoglobin molecule is called methemoglobin.
■ NADPH is rendered more efficient in the presence
of methemoglobin reductase, also called
cytochrome b5 reductase.
■ Using H1 from NADH formed when G3P is
converted to 1,3-BPG, cytochrome b5 reductase
acts as an intermediate electron carrier,
returning the oxidized ferric iron to its ferrous,
oxygen-carrying state. This enzyme accounts for
more than 65% of the methemoglobin-reducing
capacity within the RBC.
 Rapoport-Luebering Pathway
■ A third metabolic shunt generates 2,3-
bisphosphoglycerate (2,3-BPG; also called
2,3-diphosphoglycerate or 2,3-DPG).
■ 1,3-BPG diverted by bisphosphoglycerate
mutase to form 2,3-BPG.
■ 2,3-BPG binds between the globin chains in
the interior cavity of the hemoglobin tetramer
to stabilize it in the deoxygenated state (tense
or low oxygen affinity state).
■ This binding shifts the hemoglobin-oxygen
dissociation curve to the right, which
enhances delivery of oxygen to the tissues.
■ The 2,3-BPG forms 3-PG by the action of
bisphosphoglycerate phosphatase.
■ There is a delicate balance between ATP
generation to support the energy
requirements of cell metabolism and the
need to maintain the appropriate
oxygenation and deoxygenation status of
hemoglobin.
■ Acidic pH and low concentrations of 3-PG
and 2-PG inhibit the activity of
bisphosphoglycerate mutase, thus
inhibiting the shunt and retaining 1,3-BPG in
the EMP.
■ In summary, these conditions favor
generation of ATP by causing the
conversion of 1,3-BPG directly to 3-PG and
returning 2,3-BPG to 3-PG for ATP
generation downstream by PK.
RBC MEMBRANE
■ RBCs are biconcave, 7 to 8 mM in diameter, with a volume range of 80
to 100 fL and a mean volume of 90 fL. Their average surface area is 140
mm2, which is a 40% excess of surface area compared with a sphere of
7 to 8 mm in diameter. It enables RBCs to stretch undamaged up to 2.5
times.
■ RBC deformability. The RBC plasma membrane is 100 times more
elastic than a comparable latex membrane, yet it has tensile (lateral)
strength greater than that of steel.
■ The deformable RBC membrane provides the broad surface area and
close tissue contact necessary to support the delivery of O2 from the
lungs to body tissues and to transport CO2 from body tissues to the
lungs.
■ The normal mean cell hemoglobin concentration (MCHC) ranges
from 32% to 36%.
■ MCHCs greater than 36% compromise deformability and shorten
the RBC life span.

■ As the MCHC rises, the RBC, unable to pass through the splenic
pores, is phagocytized and destroyed by splenic macrophage.
RBC Membrane Lipids

■ The RBC membrane consists of approximately 8% carbohydrates, 52%

proteins, and 40% lipid.
■ The lipid portion, equal parts of cholesterol and phospholipids forms a
bilayer.
■ Phospholipids form an impenetrable fluid barrier as their hydrophilic
polar head groups are arrayed on the membrane’s surfaces, oriented
toward both the aqueous plasma and the cytoplasm, respectively, as
depicted in the fluid mosaic membrane model (FMMM).
■ Their hydrophobic nonpolar acyl tails arrange themselves to form a
central layer hidden from the aqueous plasma and cytoplasm.

■ The membrane also maintains extreme differences in osmotic

pressure, cation concentrations, and gas concentrations.
■ Cholesterol, esterified and largely hydrophobic,
resides parallel to the acyl tails of the
phospholipids.
■ With approximately one cholesterol molecule
per phospholipid molecule.
■ Cholesterol confers tensile strength to the lipid
bilayer. As cholesterol concentration rises, the
membrane gains strength but loses elasticity.
■ The ratio of cholesterol to phospholipids
remains relatively constant to maintain the
balance of deformability or elasticity and
strength.
■ Membrane enzymes maintain the cholesterol
concentration
■ The phospholipids are
asymmetrically distributed. ■ Glycolipids (sugar-bearing lipids) make
Phosphatidylcholine and up 5% of the external half of the RBC
sphingomyelin predominate in the membrane.
outer layer; ■ They associate in clumps or rafts and
■ phosphatidylserine (PS) and support carbohydrate side chains.
phosphatidylethanolamine form most
of the inner layer. ■ The glycocalyx is a layer of carbohydrates
■ Distribution of these four whose net negative charge prevents
phospholipids is energy dependent. microbial attack and mechanical damage
■ Membrane phospholipids and caused by adhesion to neighboring RBCs
cholesterol may also redistribute or to the endothelium.
laterally so that the RBC membrane
may respond to stresses and deform ■ Glycolipids may bear copies of
within 100 milliseconds by the carbohydrate-based blood group
presence of a narrow passage, such antigens.
as a capillary.

RBC Membrane Proteins
■ proteins make up 52% of the
membrane structure by mass.
■ A proteomic study revealed
there are at least 300 RBC
membrane proteins, including
105 transmembrane proteins.
Transmembrane Proteins
■ The transmembrane proteins serve many
functions including: transport sites,
adhesion sites, and signaling receptors.
■ Signaling receptors bind plasma ligands
and trigger activation of intracellular
signaling proteins, which then initiate
various energy-dependent cellular
activities, a process called signal
transduction.
■ Through glycosylation, the transmembrane
proteins also support surface
carbohydrates, which join with glycolipids
to make up the protective glycocalyx.
■ Most transmembrane proteins
assemble into one of two major
macromolecular complexes named by
their respective cytoskeletal
anchorages: the ankyrin complex and
the actin junctional complex, also
called protein 4.1 complex.
■ the linking of cytoskeletal proteins by
the actin junctional complex provides
membrane structural integrity.
■ Transmembrane proteins also provide
vertical membrane structure.
Blood group antigens

■ located in membrane macromolecular complexes that serve as

transporters, structural components, enzymes, receptors, and
adhesion molecules.
■ The carbohydrate-defined blood group antigens are supported in the
RBC membrane by transmembrane protein.
■ Band 3 (anion transport) and Glut-1 (glucose transport) support the
majority of ABH system carbohydrate determinants.
■ glycophorin A carries the peptide-defined M and N determinants and
glycophorin B carries the Ss determinants.
■ The Rh system employs two transmembrane lipoproteins and one
multipass glycoprotein, each of which crosses the membrane 12 times.
■ Additional blood group antigens localize to the actin junctional (4.1)
complex or specialized proteins.
The GPI anchor and paroxysmal nocturnal hemoglobinuria.

■ PI serves as a base on which a glycan core of sugar molecules is

synthesized, forming the glycosylphosphatidylinositol (GPI) anchor.
■ More than 30 proteins bind to the GPI anchor and appear to float on the
surface of the membrane.

■ PAROXYSMAL NOCTURNAL HEMOGLOBINURIA , an acquired mutation

in the PIGA gene affects the cells’ ability to synthesize the GPI anchor.
■ Without the GPI anchor, the cell membrane becomes deficient in CD55
and CD59, and the cells are susceptible to complement mediated
hemolysis.
 Nomenclature

■ Numerical naming, for instance, band

3, protein 4.1, and protein 4.2, derives
from historical (preproteomics)
protein identification techniques that
distinguished membrane proteins
using sodium dodecyl sulfate-
polyacrylamide gel electrophoresis
(SDS-PAGE).

■ Band 3 and protein 4.2 are the major components of the ankyrin
complex and link their associated proteins and the lipid bilayer
membrane to the spectrin cytoskeleton through ankyrin.
 Cytoskeletal Proteins Spectrin stabilization

■ The secondary structure of

■ The principal cytoskeletal
both a and b-spectrin features
proteins are the filamentous a-
triple-helical repeats of 106
spectrin and b-spectrin, which
amino acids each; 20 such
assemble to form an
repeats make up a-spectrin,
antiparallel heterodimer held
and 16 make up b-spectrin.
together with a series of lateral
■ Dematin appears to stabilize
bonds.
the actin junctional complex
and helps maintain the RBC
shape
 Membrane deformation

■ Spectrin dimer bonds that appear along

the length of the molecules disassociate
and reassociate (open and close) during
RBC deformation.
■ The lipid membrane peels off in small
fragments called blebs, or vesicles and
immediately reseals keeping the
cytoplasmic volume intact. This results in
a reduced surface area-to volume ratio
and the formation of spherocytes.
Osmotic Balance and Permeability

■ The RBC membrane is impermeable to cations Na1, K1, and Ca21.

It is permeable to water and the anions bicarbonate (HCO3-) and
chloride (Cl2), which freely exchange between plasma and RBC
cytoplasm.
■ Aquaporin 1 (Table 6.5) is a transmembrane protein that forms
pores or channels whose surface charges create inward water
flow in response to internal osmotic changes.
■ The cell swells, becomes spheroid, and eventually ruptures. This
phenomenon is called colloid osmotic hemolysis.
■ Sickle cell disease also provides an example of increased cation
permeability.
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