Assay of Brain Calmodulin Levels Using High-Performance Liquid Chromatography (original) (raw)
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Localization of calmodulin in rat cerebellum by immunoelectron microscopy
The Journal of Cell Biology, 1980
Calmodulin, a multifunctional Ca(++)-binding protein, is present in all eucaryotic cells. We have investigated the distribution of this protein in the rat cerebellum by immunoelectron microscopy using a Fab-peroxidase conjugate technique. In Purkinje and granular cell bodies, calmodulin reaction product was found localized both on free ribosomes and on those attached to rough endoplasmic reticulum (RER) and the nuclear envelope. No calmoduline was observed in the cisternae of RER or the Golgi apparactus. Calmodulin did not appear to be concentrated in the soluble fraction of the cell under the conditions used. Rather, peroxidase reaction product could be seen associated with membranes of the Golgi apparatus the smooth endoplasmic reticulum (SER), and the plasma membrane of both cell bodies and neuronal processes. In the neuronal dendrites, calmodulin appeared to be concentrated on membranes of the SER, small vesicles, and mitochondria. Also, granular calmodulin was observed in the amorphous material. In the synaptic junction, a large amount of calmodulin was seen attached to the inner surface of the postsynaptic membrane, whereas very little was observed in the presynaptic membrane or vesicles. These observations suggest that calmodulin is synthesized on ribosomes and discharged into the cytosol, and that it then becomes associated with a variety of intracellular membranes. Calmodulin also seems to be transported via neuronal processes to the postsynaptic membrane. Calmodulin localization at the postsynaptic membrane suggests that this protein may mediate calcium effects at the synaptic junction and, thus, may play a role in the regulation of neurotransmission.
Presence of Calmodulin and Calmodulin-Binding Proteins in the Nuclei of Brain Cells
Journal of Neurochemistry, 1991
The nuclear calmodulin levels have been measured in rat neurons and glial cells. The values are 1 .O and 1.1 pg/ mg of protein, respectively. These levels are about threefold higher than those in the nuclei of rat liver cells. We have also investigated the presence of several calmodulin-binding proteins in the nuclei of both brain cellular types. As similarly observed in the nuclei of liver cells, we detected the presence of a-spectrin and a 62-kDa calmodulin-binding protein in the nuclei of neurons and glial cells by immunoblotting and immunocytochemical methods. Both proteins are enriched in the purified nuclear matrix samples from both cel-lular types. In contrast to that occurring in rat hepatocytes, we have not been able to detect, by immunoblotting methods, caldesmon in the nuclear matrices of neurons and glial cells. The immunocytochemical studies suggest, however, that caldesmon can be present in the nuclei but in a fraction distinct from the nuclear matrices. Key Words: Calmodulin-Calmodulin-binding proteins-Caldesmon-a-Spectrin-Brain cortex-Neuronal and glial cell nuclei. Vendrell M. et al.
Synapse, 1990
Calmodulin is a small, acidic calcium-binding protein that regulates a number of calcium-dependent enzyme activities and is thought to be involved in neurotransmission. To begin to explore further the regulation of this important protein in the brain, we have cloned a rat calmodulin cDNA and designed an oligonucleotide probe based on this sequence. Both the cDNA and oligonucleotide probes revealed a markedly heterogeneous distribution of hybridization signal for calmodulin mRNA in the rat brain. The greatest apparent abundance of mRNA for calmodulin was seen in the hippocampus and cerebral cortex, whereas many brain regions showed relatively low hybridization signal, including the striatum and portions of the hypothalamus and brainstem.
Calmodulin is a potent target for new hypothalamic neuropeptides
FEBS Letters, 1990
Recently, five glycopeptides with coronaro-constrictory properties were isolated from bovine hypothalamus [(1988) Neurochemistry (USSR) 7,519-5241. Calmodulin has been recognized in our laboratory as a target protein for the neuropeptides isolated from hypothalamus. The results of indirect enzyme-linked immunosorbent assay have shown that the new hypothalamic neuropeptides antagonize with the monospecific anti-calmodulin antibody for calmodulin binding although they are not fragments of calmodulin. The inhibitory potency of the peptides is dependent on their concentration and the length of the polypeptide chain. Four out of five peptides are effective in nM concentration range. Ca*+ stimulates the binding of peptides to calmodulin; however, immunocomplex can be formed in the absence of Ca*+ as well. The effects of trifluoperazine and peptides on the calmodulin/antibody interaction are not additive, suggesting the cooperativity between the binding sites on calmodulin. Under physiological conditions the presence of the peptides could produce distinct conformers of calmodulin which may exhibit altered potency for stimulation/inhibition of target enzymes. Anti-CaM activity; Neuropeptide; Enzyme-linked immunosorbent assay; Cooperative binding
[ 125 I]calmodulin binding to synaptic plasma membrane from rat brain: Kinetic and arrheniu
Neurochem Res, 1993
Binding of [t25I]calmodulin was characterized in highly purified synaptic plasma membrane (SPM) prepared from rat brain. By Scatchard analysis, the CaZ+-dependent membrane binding of [lzSI]caImodulin was found to have a Bmax of 284 pmo1/mg protein and an apparent affinity with a I% of 131 riM. Kinetic analysis indicates that at 37 ~ the dissociation of [125I]calmodulinmembrane complexes follows first-order reaction and consists of two components: a dissociation constant (k) of 3.7 x 10 -1 min -1 and a half-time (t~,~) of 1.8 min for the fast component, and a k of 4.8 x 10 -2 rain -~ and a t~ of 14.5 min for the slow component. At 0 ~ substantial dissociation still occurred, with a k of 4.5 x 10 -2 min -~ and a t~/2 of 15.3 min for the fast component, and a k of 5.5 x 10 -3 min -1 and a t~,~ of 125.5 min for the slow component. These data on binding affinity and dissociation kinetics are consistent with the notion that SPM can readily and rapidly associate and dissociate calmodulin. In Arrhenius analysis of temperature effects, [z2sI]calmodulin binding to SPM exhibits a biphasic function, with the transition temperature (Td) estimated to be 23.8~ suggesting that binding is influenced by lipid phase transition of the membrane. The binding of [~2SI]calmodulin to the synaptic membrane was found to be increased by corticosterone (10 -7 -10 -6 M), a steroid hormone, and decreased by ethanol (50-200 raM), a centrally acting drug. Our data on the characteristics of calmodulin binding to the SPM provide groundwork for future studies on physiological and pharmacological regulation of calmodulin translocation to and from the plasma membrane in synaptic terminals.
[125I]calmodulin binding to synaptic plasma membrane from rat brain: Kinetic and arrhenius analysis
Neurochemical Research, 1993
Binding of [t25I]calmodulin was characterized in highly purified synaptic plasma membrane (SPM) prepared from rat brain. By Scatchard analysis, the CaZ+-dependent membrane binding of [lzSI]caImodulin was found to have a Bmax of 284 pmo1/mg protein and an apparent affinity with a I% of 131 riM. Kinetic analysis indicates that at 37 ~ the dissociation of [125I]calmodulinmembrane complexes follows first-order reaction and consists of two components: a dissociation constant (k) of 3.7 x 10 -1 min -1 and a half-time (t~,~) of 1.8 min for the fast component, and a k of 4.8 x 10 -2 rain -~ and a t~ of 14.5 min for the slow component. At 0 ~ substantial dissociation still occurred, with a k of 4.5 x 10 -2 min -~ and a t~/2 of 15.3 min for the fast component, and a k of 5.5 x 10 -3 min -1 and a t~,~ of 125.5 min for the slow component. These data on binding affinity and dissociation kinetics are consistent with the notion that SPM can readily and rapidly associate and dissociate calmodulin. In Arrhenius analysis of temperature effects, [z2sI]calmodulin binding to SPM exhibits a biphasic function, with the transition temperature (Td) estimated to be 23.8~ suggesting that binding is influenced by lipid phase transition of the membrane. The binding of [~2SI]calmodulin to the synaptic membrane was found to be increased by corticosterone (10 -7 -10 -6 M), a steroid hormone, and decreased by ethanol (50-200 raM), a centrally acting drug. Our data on the characteristics of calmodulin binding to the SPM provide groundwork for future studies on physiological and pharmacological regulation of calmodulin translocation to and from the plasma membrane in synaptic terminals.
Neurochemical research, 1998
Calmodulin (CaM) through activation of CaM-kinase II may be involved in the molecular mechanisms underlying the epileptogenic processes. Some evidence suggests that kindling responses change across the day-night cycle. In order to test if kindling stimulation modifies CaM content, we measured CaM concentrations in amygdala, hippocampus and hypothalamus obtained from control and kindled rats during light and darkness. Male Wistar rats (250-300 g), were injected i.p. with Pentylenetetrazol (PTZ) (35 mg/kg/24 h). Once chemical kindling was established, rats were sacrificed by decapitation at 10:30 a.m. and 01:30 a.m. The brains were obtained, and the amygdala, hippocampus and hypothalamus dissected. CaM content was measured in the cytosol and membrane fractions by radioimmunoassay. We found a significant increase in CaM content in cytosol and membrane fractions of both control and kindled rats during the dark phase. No significant differences in CaM concentrations were observed between...