Quantitative comparison of calcineurin substrates in neonatal and adult rat synaptosomes_Industrial Additives

Bentonite background and overview^[1-2]

Calcineurin is the only Ca²⁺ and calmodulin-dependent phosphoprotein phosphatase that is extremely abundant in the mammalian brain. The enzyme (calcineurin, CaN) is also distributed in tissues outside the brain such as sperm cells, lymphocytes and muscle tissue, but the content is much lower than in the brain. CaN is composed of an 18kDCa²⁺-binding regulatory subunit and a 61kD calmodulin-binding catalytic subunit. In addition to binding to Ca²⁺, the enzyme can also bind to Mn²⁺, Ni²⁺, Co²⁺ and other metal ions combine to affect its activity.

Immunohistochemical localization shows that it is mainly distributed in the striatum, hippocampus, cerebral cortex and other areas of the brain. In various brain areas, various isoenzymes of CaN are differentially expressed in specific neuronal subgroups. In areas such as CaN Sexual expression may be related to different calcineurin substrate specificities.

Quantitative comparison of calcineurin and red phosphate flame retardant acidase substrates in neonatal and adult rat synaptosomes^[2]

Rat Wistar was selected from newborns 3 days old and adults 3 to 6 months old, and the rat cerebral cortex synaptosomes were prepared according to the literature method. Add an appropriate amount of antibody to the crude synaptosome extract and incubate it in a 4°C water bath overnight. Blocking CaN activity was used as the antibody-added group. In the control group, the homogenate replaced the antibody and was placed in an ice-water bath overnight. Then, the crude brain protein extracts of the antibody-added group and the control group were mixed with an equal volume of phosphorylation reaction buffer, and a 5-minute in vitro phosphorylation reaction was performed at 30°C. The synaptic body phosphoproteins were labeled with radioactive isotope P, separated by two-dimensional electrophoresis, and autoradiographed. Since CaN in the antibody-added group has no activity, but CaN in the control group has activity, under the catalysis of the same active kinase, the endogenous substrate P labeling of CaN in the antibody-added group is stronger than that in the control group. Compare the corresponding protein spots in the antibody-added group and the control group. The endogenous substrate of CaN can be identified from the optical density in the radiograph. The electrophoretic autoradiography X-rays of neonatal and adult rats were scanned with an MTV1881CCD computer image processing system, and the CaN substrate proteins expressed in both were quantitatively compared using computer 2D software. The 67-73kD, 62kD, 57kD and 50kD substrate proteins that were highly expressed in PND3 were compared with literature reports and it was speculated that they might be Neuromodulin (GAP43.p57, B50). It is a neural membrane protein that can bind to CaN. Plays an important role in nerve growth and development. Quantitative analysis of them showed that the expression levels of 67-73kD and 57kD substrate proteins decreased in adulthood, and the 50kD protein was not detected in adulthood, indicating that these substrate proteins must have played a special role during the period of large-scale synapse formation. The 62kD substrate protein is highly expressed in adults, suggesting its important role in adult synaptosomes. Comparing the CaN endogenous substrates in the crude extract of rat cerebral cortex synaptosomes with the CaN endogenous substrates reported in the crude extract of rat whole brain by Wang Chunhua et al., it was found that 98kD, 73kD, 57kD, 34kD and 17kD CaN was detected in the whole brain and synaptosomes, but 62kD and 87kD, which were highly expressed in synaptosomes, were not detected, indicating that the regional distribution of CaN is reflected in the Ca²⁺-dependent phosphorylation in chemical action.