A new spectrophotometric assay for 2′,3′-cyclic nucleotide 3′-phosphohydrolase activity in nervous tissue

Dreiling, Charles E.; Mattson, Cynthia

doi:10.1016/0003-2697(80)90157-8

Cited by 14 publications

(2 citation statements)

References 17 publications

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“…The suspension was layered on a step gradient consisting of the following sucrose solutions in 5 mM Hepes (pH 7.4): 2 ml, 1.5 M; 10 ml, 1.2 M; 6 ml, 1.0 M; 7 ml, 0.8 M. After centrifugation in a Beckman SW 25.1 rotor at 90,000 x g for 2 hr, 2.3-ml fractions were collected. Fractions enriched in myelin, synaptosomes, and mitochondria were identified by the use of appropriate enzyme markers (12,13). The fractions were diluted to isotonicity with appropriate volumes of ice-cold 5 mM Hepes (pH 7.4) and centrifuged at 12,000 x g for 30 min.…”

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confidence: 99%

Calcium/phospholipid regulates phosphorylation of a Mr "87k" substrate protein in brain synaptosomes.

Wu¹,

Walaas²,

Nairn³

et al. 1982

Proc. Natl. Acad. Sci. U.S.A.

217

107

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Depolarization-induced calcium influx into rat cerebral cortex synaptosomes increased the phosphorylation of several synaptosomal proteins as examined by 32Pi incorporation. A phosphopeptide mapping technique involving NaDodSO4/ polyacrylamide gels has been used to show that phosphorylation of a Mr 87,000 substrate protein is stimulated by depolarizationinduced calcium influx. Phosphorylation of this Mr 87,000 substrate occurred in synaptosomal cytosol and was markedly stimulated by calcium/phosphatidylserine. Calmodulin inhibited this phosphorylation reaction. This substrate for calcium/phospholipid-dependent protein kinase is enriched in and appears to be specific to neurons.Calcium is recognized to play a key role in the regulation of neurotransmitter release from nerve endings (1). Increasing evidence indicates that some of the intracellular actions of calcium in neurons may be mediated through protein phosphorylation (see review in ref. 2). By using fractionated nerve endings (synaptosomes) from the brain, it was demonstrated that calcium influx induced by membrane depolarization resulted in increased phosphorylation of specific intraterminal proteins, the most prominent of which was protein I (3). Activation by calcium of the phosphorylation of protein I and of certain other synaptosomal proteins has been shown to be mediated by calmodulin-dependent protein kinases (4-6).Recently, a second species of calcium-dependent protein kinase has been described that requires phospholipid (7) rather than calmodulin for its activity. This enzyme occurs at a relatively high level in mammalian brain (8). We report here the occurrence of a Mr 87,000 phosphoprotein (termed the 87k protein) in the nerve terminals from rat cerebral cortex; the phosphorylation of this protein is regulated by Ca2' influx through activation of a Ca2+/phospholipid-dependent protein kinase.MATERIALS AND METHODS Materials. Bovine brain L-a-phosphatidyl-l-serine (98-99% pure) and diolein (99%) were purchased from Sigma. Calmodulin was purified from rabbit brain according to the procedure of Grand et al. (9). Standard protein I was purified from bovine brain by a modification (unpublished results) of the original procedure (10). The catalytic subunit of cAMP-dependent protein kinase was purified from bovine heart as described (11).Preparation of Synaptosomes. A crude mitochondrial fraction (P2) containing synaptosomes was prepared as described (3). For further subfractionation, the P2 pellet prepared from three rats was suspended in 5 ml of 0.32 M sucrose/5 mM Hepes, pH 7.4, at a protein concentration of 8-10 mg/ml. The suspension was layered on a step gradient consisting of the following sucrose solutions in 5 mM Hepes (pH 7.4): 2 ml, 1.5 M; 10 ml, 1.2 M; 6 ml, 1.0 M; 7 ml, 0.8 M. After centrifugation in a Beckman SW 25.1 rotor at 90,000 x g for 2 hr, 2.3-ml fractions were collected. Fractions enriched in myelin, synaptosomes, and mitochondria were identified by the use of appropriate enzyme markers (12,13). The fractions were diluted to ...

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mentioning

confidence: 99%

Calcium/phospholipid regulates phosphorylation of a Mr "87k" substrate protein in brain synaptosomes.

Wu¹,

Walaas²,

Nairn³

et al. 1982

Proc. Natl. Acad. Sci. U.S.A.

217

107

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“…This reaction can be used to test for the presence or absence of functional CNPase enzyme secreted by the cells (Figure 1). The methodology used was adapted from Dreiling and Mattson [1980], where CNPase + 2'3'-cAMP + Water + phenol red = 2'AMP + H + causing a decrease in pH that can be detected using a colourimetric assay (32) CNPase activity increases, pH will decrease (32), and when measuring that pH change with phenol red at 560 nm, it will result in a drop in absorbance (33).…”

Section: Cnpase Enzyme Assaymentioning

confidence: 99%

Neurogenic marker expression in differentiating human adipose derived adult mesenchymal stem cells

Pelegri¹,

Milthorpe²,

Gorrie³

et al. 2023

Stem Cell Investig

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Background: Adipose-derived stem cells (ADSCs) are increasingly utilised in the field of neural regeneration due to their high accessibility and capacity for differentiation into neural like cells. Culturing ADSCs in the presence of various growth factors, small molecules and combinations thereof have shown promise in this regard; however, these protocols are generally complex, time-consuming and costly. The need for commercially available and chemically defined growth media/supplements is required to facilitate further developments in this area.Methods: In this study, we have examined the neural differentiation and proliferation potential of the commercially available supplements B27, CultureOne (C1) and N2 on human ADSCs (hADSCs). Through a combination of immunocytochemistry, cytokine analysis, and CNPase enzymatic assays, we provide novel insight into the neural differentiation effects of B27, C1 and N2 on hADSCs. Results:The study found that C1 and N2 supplements initiated neural differentiation of the cells, with C1 pushing differentiation towards an oligodendrocytic lineage and N2 initiating neuronal differentiation. This suggests that C1 and N2 supplements can be used to drive neural differentiation in hADSCs. However, B27 did not show significant differentiation in the time frame in which the experiments took place and therefore is unsuitable for this purpose.Conclusions: These findings highlight the utility of commercially available supplements in the neural differentiation of ADSCs and may assist in establishing simpler, more affordable differentiation protocols.

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Myelin deposition in the optic tectum of trout as monitored by enzymatic and morphometric analyses

Jeserich

Breer

1981

Neurochem Res

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The activity of arylsulfatase A and 2'3'-cyclic nucleotide 3'-phosphohydrolase was studied in the brain of trout in parallel to the structural differentiation of tissue from early larval stages into adulthood. Whereas in the optic tectum, phosphodiesterase activity could not be detected before the second month after hatching in brainstem, the enzyme had already reached 80% of adult level. In tectum it was from the fourth to the seventh month that this enzyme dramatically increased, thereby reaching about the adult level. The developmental profile of arylsulfatase A was profoundly different, since 1) considerable activity was found in tectum at early larval stages and 2) the activity showed a peak between two and six months and then dropped markedly. Morphometric analysis of the two myelinated layers of trout tectum support and extend the biochemical results leading to the conclusion that the phosphodiesterase activity reflects the prevailing degree of myelination, whereas the developmental profile of the sulfolipid-metabolizing enzyme indicates the rate of myelin accumulation.

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A new spectrophotometric assay for 2′,3′-cyclic nucleotide 3′-phosphohydrolase activity in nervous tissue

Cited by 14 publications

References 17 publications

Calcium/phospholipid regulates phosphorylation of a Mr "87k" substrate protein in brain synaptosomes.

Calcium/phospholipid regulates phosphorylation of a Mr "87k" substrate protein in brain synaptosomes.

Neurogenic marker expression in differentiating human adipose derived adult mesenchymal stem cells

Myelin deposition in the optic tectum of trout as monitored by enzymatic and morphometric analyses

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