Cell and Tissue Journal

Abstract Aim: Red blood cells and white blood cells are the main  cells of blood.These two types of cells have significant differences in number, nucleus, and other intracellular organelles such as mitochondria, ribosomes, and metabolic pathways. Enzymes are important proteins found in these cells . MAPKinase (Mitogen activated protein kinase) superfamily are protein kinases playing key role in phosphorylation of threonine , thyrosine and serine in the enzymes of the family and target proteins during kinase cascades  in metabolic pathways  of cells. They are found in nucleated cells from unicellular to multicellular. These enzymes have important roles in regulating various processes of eukaryotic cells, including cell proliferation, differentiation, survival , apoptosis  and  in various signaling pathways and gene expression as well. These enzymes are ubiquitously expressed and evolutionarily conserved in eukaryotes. In mammalian cells, there are three well-known MAPK   including  extracellular signal-regulated protein kinase (ERK) 1/2 , c-Jun N-terminal Kinase 1, 2, 3 (JNK1/2/3) and p38 MAPK α,β,δ,γ
ERK, JNK and p38 isoforms are grouped according to their motif, structure and function.  ERK 1/2 is related to  the response to growth factors, hormones and inflammatory stimuli, while JNK1/2/3 and p38 MAPK α, β, δ, and γ are activated through environmental or cellular stress and inflammatory stimuli.
JNK   enzyme is activated under stress. JNK pathway has role in apoptosis and cell survival. The presence of JNK is essential for stress-induced mitochondrial cytochrome c release. Cytochrome c together with Apaf activates the initiator caspase 9. If the defect in the release of cytochrome c from the mitochondrial membrane is likely to cause a defect in the release of pro-apoptotic molecules such Smac/DIABLO, AIF. The aim of the study is to investigate the JNK enzyme level in blood anucleated cell such as RBC compared to nucleated cell like WBC. 
Materials and Methods: RBCs were isolated from a fresh blood. WBCs (Mononuclear) were separated from blood using Ficoll solution.  Their suspensions were prepared in isotonic condition using 0.9 % NaCl. In next step, the number of cells counted by means of cell counter followed by lysis  RBCs by ultrasonic  homogenizer and  lysis of WBCs using  ultrasonic bath. Considering that, the presence of hemoglobin following the lysis of RBCs affects the assay of JNK level by ELISA immunoassay technique, hence 6 mM zinc sulfate used to remove the hemoglobin. Two kinds of lysates were centrifuged to separate the  lysed cells membranes before assay the level of JNK.  Then, the level of JNK in the RBC and WBC lysates were measured using ELISA technique.
Results: Regarding that the number of RBCs in sample was 1000 times more than WBCs one per sample volume, but the JNK enzyme level showing  1.72 x10 ^-2  ng/ml per cell and 6.2 x 10 ^-6  ng/ml per cell in WBCs and RBCs  respectively. As a result, JNK enzyme level in each WBC   is 2770 fold   more than each RBC.
Conclusion: In comparison with WBCs having nuclei and high level of   JNK enzyme,  RBCs due to losing their nuclei during differentiation from stem cells in bone marrow show low level of JNK enzyme denoting blocking of pathways related to MAPK enzymes. This is an evidence that the absence of nucleus does not support of MAPK family enzyme and related pathways.