Purification and immunohistochemical analysis of calcium-binding proteins expressed in the chick pineal gland

Department of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, Tokyo 113-0033, Japan.
Journal of Pineal Research (Impact Factor: 9.6). 05/2003; 34(3):208-16. DOI: 10.1034/j.1600-079X.2003.00031.x
Source: PubMed


The pineal gland is a site of melatonin production, of which intracellular calcium ions (Ca2+) are likely involved in various aspects. To investigate the identity of molecules responsible for the Ca2+-dependent processes in the pineal cells, we prepared a cellular extract from 2000 chick pineal glands and isolated a series of Ca2+-binding proteins by taking advantage of their Ca2+-dependent hydrophobic interaction with phenyl-Sepharose beads. The proteins identified by micro-sequencing analysis included calmodulin, neurocalcin, sorcin, annexin II and annexin V. Immunohistochemical analysis of the chick pineal sections revealed that both calmodulin and sorcin are expressed in the follicular and parafollicular pinealocytes. On the other hand, neurocalcin was expressed in a few neuron-like cells located predominantly in the parafollicular layer of the pineal follicle. These results suggest that calmodulin and sorcin may contribute to cellular functions in the chick pinealocytes.

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    Frontiers in Microbiology 02/2014; 5:63. DOI:10.3389/fmicb.2014.00063 · 3.99 Impact Factor
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    • "Each pool of the frozen glands was homogenized separately with 200 lL of sodium dodecyl sulfate–polyacrylamide gel electrophoresis loading buffer containing 20 mmol/L NaH 2 PO 4 , 10 mmol/L Na 2 H 2 P 2 O 7 , 1.6 mmol/L EDTA, and 1.6 mmol/L EGTA, and the total protein concentration was estimated by the method of Bradford (1976) to normalize sample-to-sample variation. Equal amounts of proteins were loaded onto an 8.5% polyacrylamide gel and were subjected to immunoblotting as described previously (Shimizu et al. 2003). "
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