RED BLOOD CELLS

Extracts of rabbit reticulocytes (obtained by injecting rabbits with a chemical-phenyl hydrazine- that causes a severe hemolytic anemia, so that the red cells are almost completely replaced by reticulocytes.) are widely used in in-vitro protein synthesis.

Endogenous mRNAs present are destroyed by nuclease; nuclease activity inhibited by addition of calcium ions.

The radioactive proteins synthesized are separated by SDS-PAGE and detected by auto-radiography.

In vertebrates, erythrocyte width is on average about 25% larger than “capillary diameter”. The hypothesis is – this improves the oxygen transfer from erythrocytes to tissues.

It has been recently demonstrated that erythrocytes can also synthesize “NO” enzymatically using L-arginine as substrate, just like ‘endothelial cells”. Exposure of erythrocytes to physiological levels of shear stress activates- NITRIC OXIDE, which may contribute to the regulation of vascular tissue/ tonus.

Erythrocytes can also produce “hydrogen sulfide, a signalling gas that acts to relax vessels. It is believed that the cardio-protective effects of “garlic” are due to erythrocytes converting its sulfur compounds into hydrogen sulfide gas.

Vitamin C aids iron absorption.

RBC AS ANTIGEN:RBC contains exogenous mannose that helps them evade immune response.

RBC: PHYSIOLOGY

Pentose phosphate pathway* and glutathione peroxidase protect the erythrocyte against hemolysis. They provide NADPH for the reduction of oxidized glutathione catalyzed by glutathione reductase, a flavoprotein containing FAD.Reduced glutathione removes hydrogen peroxide in a reaction catalyzed by glutathione-peroxidase; an enzyme that contains the selenium analogue of cysteine or selenocysteine as its active site.

This reaction is important, since accumulation of hydrogen peroxide may decrease the life-span of the erythrocyte by causing oxidative damage to the cell-membrane, leading to hemolysis.

Impairment of pentose phosphate pathway by sulfonamides, anti-malarial primaquine fava-beans etc. This impairment leads to hemolysis or hemolytic malaria.

*Pentose phosphate pathway is active in liver, adipose tissue, adrenal cortex, thyroid, erythrocytes, testis and non-lactating mammary glands(?) and skeletal muscle.

How does pentose phosphate pathway protect RBC against hemolysis?

GENERATION OF BI-CARBONATE IONS BY RBC

Due to lack of aerobic metabolic pathways, RBC produce very little carbon-dioxide.The plasma carbon-dioxide diffuses into the RBC along the concentration gradient where it combines with water to form carbonic acid. This reaction is catalysed by carbonic anhydrase( also known as carbonate dehydratase). In the RBC, carbonic acid dissociates to produce H+ and HCO3

-.H+ ions are trapped and buffered by hemoglobin.When HCO3

- increases, it diffuses out of RBC to plasma, along with the concentration gradient, in exchange for Cl- ions to maintain electrical neutrality. This phenomenon, referred to as chloride shift, helps to generate bicarbonate ions.

RESPIRATION IN RBC:GLUCOSE TRANSPORT-Instead of insulin mediated uptake of glucose, RBC follows an insulin independent transport System.Glucose transporter GLUT 1 is present in erythrocytes.Glycolysis is the major pathway for ATP synthesis in tissues lacking mitochondria e.g. erythrocytes, cornea, lens etc.

SPECIALISED PATHWAY IN ERYTHROCYTES-

A supplementary pathway called RAPAPORT-LEUBERING CYCLE, operates in erythrocytes of man and other mammals.2, 3- bisphospho glycerate synthesis in RBC ,instead of 1,3- bisphosphate synthesis.Anerobic glycolysis leads to lactic acid production; out of which 15 to 25 % glucose follow the above mentioned pathway.The functionality of this pathway is to dissipate or waste the energy not needed.More over, it aids in unloading more oxygen to the tissues.

Proof that polypeptides grow by addition of aminoacid residues to the carboxyl end: the Dintzis experiment. Reticulocytes(immature erythrocytes) actively synthesizing hemoglobin wereincubated with radioactive leucine (selected because it occurs frequentlyin both the alpha- and beta-globin chains). Samples of completed _chains were isolated from the reticulocytes at various times afterward,and the distribution of radioactivity was determined. The dark redzones show the portions of completed alpha-globin chains containing radioactiveLeu residues. At 4 min, only a few residues at the carboxylend of alpha-globin were labeled, because the only complete globin chainswith incorporated label after 4 min were those that had nearly completedsynthesis at the time the label was added. With longer incubationtimes, successively longer segments of the polypeptide containedlabeled residues, always in a block at the carboxyl end of thechain. The unlabeled end of the polypeptide (the amino terminus) wasthus defined as the initiating end, which means that polypeptides growby successive addition of amino acids to the carboxyl end.

Some proteins contain covalent disulfide (S-S) bonds that link the sulfhydryl groups of cysteinyl residues.Formation of disulfide bonds involves oxidation of the cysteinyl residues .Formation of disulfide bonds involves oxidation of the cysteinyl sulfhydryl groups and requires oxygen.Glutathione can reduce inappropriate disulfide bonds that may be formed upon exposure to oxidizing agents such as 02, hydrogen peroxide or superoxide.

RBC RESPONSE TO ENVIRONMENTAL THREAT-Ferritin, an iron binding protein, prevents ionized iron Fe3+ from reaching toxic levels within cells. Elemental iron stimulates ferritin synthesis by causing the release of a cytoplasmic protein that binds to a specific region in the 5’ non-translated region of ferritin mRNA. Disruption of this protein-mRNA interaction activates ferritin mRNA. Disruption of this protein-mRNA interaction activates ferritin mRNA and results in its translation. This results in its translation. This mechanism provides for rapid control of the synthesis of a protein that sequesters Fe2+, a potentially toxic molecule.

RBC PLEOMORPHISM-The red blood cell must be able to squeeze through some tight spots in the micro-circulation during its numerous passages around the body; the sinusoids of the spleen are of special importance in this regard. For the red blood cells to be easily and reversible deformable, its membrane must be fluid as well as flexible; it should also preserve its bi-concave shape; since this facilitates gas exchange. Membrane lipids help determine the membrane fluidity. Attached to inner aspect of the membrane of the red blood cell are a number of peripheral cytoskeletal proteins that play important roles in respect to preserving the shape and flexibility.

FUSION CAPACITY OF RED BLOOD CELLS

On energy depletion, RBC undergo suicidal cell death oreryptosis characterized by cell swelling and phosphatidylserine exposure at their surface

Recent observations have revealed a role for AMPK in the regulation of eryptosis duringenergy depletion.

AMPK1 is important for red cell membrane deformability

Putative AMPK substrate proteins in RBC were recently identified [12], including the band 3protein from the red cell membrane and enzymes involved in the oxidative stress response,which may contribute to the molecular defects associated with the increased membranerigidity of AMPK α1-/- RBC [7]

In conclusion, impaired function of AMPK1 induces sufficient alterations in RBCstructural flexibility to affect their lifespan and to incurably provoke their elimination fromcirculation.

REFERENCES-1)Weil, P.Anthony, Harper’s illustrated biochemistry Chapter 37 PROTEIN SYNTHESIS AND THE GENETIC CODE.2)Kamal.J.K Amisha, Behere.Digamber.V, “BINDING OF HEME TO HUMAN SERUM HEMOGLOBIN: STEADY-STATE FLUOROSCENCE, CIRCULAR DICHROISM AND OPTICAL DIFFERENCE SPECTROSCOPIC STUDIES”; Indian journal of Biochemistry and Biophysics; Vol.42, February 2005; pg-7-12