Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.11.17 (CaMKII)
 4,029 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

CaM kinase II is known to be enriched in mammalian and avian brains. To determine the holoenzymic composition and functional characteristics of this kinase, a new approach for isolation was applied to isolate it from the chicken forebrain. Forebrains of hatched 45-d chicken were dissected, homogenized, and centrifuged. The supernatant was loaded onto a CaM-agarose affinity column and the calmodulin-binding proteins were eluted with EGTA. Selected eluates were loaded onto the antibody-agarose affinity column, which was prepared with monoclonal antibody (MAb) (6G9) to the CaM kinase II alpha subunit. Samples were subjected to SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and either silver-stained or blotted onto a nitrocellulose membrane. The protein composition and the immunoreactivity of the antibody-agarose affinity eluate fractions were analyzed with a densitometric scanner. Silver staining of gels showed that the beta subunit doublet, the beta' subunit, and a putative substrate were coeluted with the alpha subunit from the antibody affinity column although only the alpha subunit bound the 6G9 antibody. Scintillation counting showed that the autophosphorylation of the kinase was significantly reduced in the eluate from the antibody affinity column. Whereas silver staining indicated an increase in the relative amount of alpha subunit had occurred during purification, phosphorylation assays indicated an increase in the relative amount of the alpha subunit after the last purification step. A possible reason for this is discussed. The presence of beta/beta' subunits in the antibody-agarose affinity eluate indicated the existence of an alpha beta/beta' heteropolymer. The phosphorylation assay was not a good indication of the amount of purification because of the loss of enzyme activity following purification. In contrast, the immunoassay indicated a 97-fold purification from the cytosolic fraction was achieved using the method. In conclusion, the data indicate the existence of the CaM kinase II alpha beta/beta' heteropolymer in the chicken forebrain.
 ...
PMID:Purification and characterization of the Ca2+/calmodulin-dependent protein kinase II from chicken forebrain. 765 21

Retinal cytosolic Ca2+/calmodulin-dependent protein kinase II (CaM KII) was isolated from hatched 6-wk chicken retinae by ultracentrifugation and affinity chromatography using calmodulin (CaM) and anti-CaM KII-alpha columns. Samples from different fractions were examined with SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and silver staining or immunoblotting. Comparisons were made between the final antibody affinity eluates from retina and forebrain. Silver-stained gels showed that multiple proteins were present in the antibody affinity eluates from retina, including major proteins of 178, 56, and 45 kDa and several minor proteins. Immunoblots revealed that CaM KII-alpha was present in eluates from the retina and forebrain. CaM KII-beta was present in the antibody eluate from forebrain but not retina. The latter subunit was present in the crude homogenates of the retina. Regarding the antibody eluate from retina, the possibility that the major 56 kDa protein was tubulin was ruled out, but protein tau (tau) and synapsin I were present. The presence of multiple proteins in the antibody affinity eluate indicates that these proteins were coisolated in a CaM KII-alpha-associated protein complex. The finding that protein tau and synapsin I are associated with retinal CaM KII provides further insight into the mechanisms underlying the function of the kinase in this tissue. The lack of cytosolic CaM KII-beta subunit in the antibody affinity eluate from retina is indicative of a brain region-specificity in subunit composition of the kinase.
 ...
PMID:The Ca2+/calmodulin-dependent protein kinase II-associated protein complex isolated from chicken retina. 883 78

Cardiac hypertrophy occurs in as many as 47% of normotensive individuals who chronically use cocaine. We investigated the effects of cocaine, in concentrations commonly found in chronic cocaine users, on calcium/calmodulin kinase (CaMK), and whether cocaine can activate CaMK, increase cardiac myocyte protein expression, and cause cardiac hypertrophy in this manner. In series I to III, 0 (control) or cocaine in concentrations of 10 to 10 mol/L was added to cultured adult rat cardiac ventricular myocytes to determine by Western blots and by P incorporation the optimal treatment time and the optimal dose for CaMK activation. In series I, cocaine, 10 mol/L, increased myocyte CaMKII translocation from myocyte soluble to particulate fractions by > or =73 +/- 9% (P < 0.01) in comparison with controls but did not cause the translocation of CaMKI or CaMKIV. In series II and III, cocaine treatment of myocytes for 15 minutes increased maximal CaMKII activity by 86.5 +/- 13.3% (P < 0.001) and a cocaine dose of 5 x 10 mol/L increased CaMKII activity by 169.5 +/- 18.1% (P < 0.001). In series IV we measured by silver staining beta-myosin heavy chain protein (beta-MHC) expression in myocytes before and after cocaine and also CaMK inhibition with KN-62 (1-[N,O-bis-(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine). In these experiments, cocaine, 5x10 mol/L, increased myocyte protein concentration by 29.2 +/- 2.8%, and beta-MHC by 93.2 +/- 8.8% (P < 0.001). In series V and VI, cocaine effects on calcium currents (ICa) and intracellular Ca ([Ca]i) were determined before and after CaMK inhibition with KN-62 in rat myocytes. Cocaine, 10 mol/L, enhanced ICa peak amplitude in a voltage-dependent manner (by 173.9 +/- 14.9% at -20 mV and by 38.4 +/- 6.9% at 0 mV P < 0.01). Cocaine, 10 to 10 mol/L, in series VI promoted Ca transients from myocyte sarcoplasmic reticulum and increased [Ca]i to 607 +/- 141 x 10 mol/L (P < 0.05). KN-62 decreased cocaine-induced myocyte protein expression by 76.6%, and beta-MHC by 66.2% (P < 0.01) and significantly decreased cocaine-induced Ca transients and [Ca]i. We conclude that CaMKII activation is an important mechanism whereby cocaine can cause myocyte hypertrophy.
 ...
PMID:Cocaine activates calcium/calmodulin kinase II and causes cardiomyocyte hypertrophy. 1689 8

Synapsins are synaptic vesicle-associated phosphoproteins that play a major role in the fine regulation of neurotransmitter release. In Drosophila, synapsins are required for complex behavior including learning and memory. Synapsin isoforms were immunoprecipitated from homogenates of wild-type Drosophila heads using monoclonal antibody 3C11. Synapsin null mutants (Syn(97)) served as negative controls. The eluted proteins were separated by SDS-PAGE and visualized by silver staining. Gel pieces picked from five bands specific for wild type were analyzed by nano-LC-ESI-MS/MS following multienzyme digestion (trypsin, chymotrypsin, AspN, subtilisin, pepsin, and proteinase K). The protein was unambiguously identified with high sequence coverage (90.83%). A number of sequence conflicts were observed and the N-terminal amino acid was identified as methionine rather than leucine expected from the cDNA sequence. Several peptides from the larger isoform demonstrated that the in-frame UAG stop codon at position 582 which separates two large open reading frames is read through by tRNAs for lysine. Seven novel phosphorylation sites in Drosophila synapsin were identified at Thr-86, Ser-87, Ser-464, Thr-466, Ser-538, Ser-961, and Tyr-982 and verified by phosphatase treatment. No phosphorylation was observed at the conserved PKA/CaM kinase-I/IV site (RRFS, edited to RGFS) in domain A or a potential PKA site near domain E.
 ...
PMID:Mass spectrometric analysis of synapsins in Drosophila melanogaster and identification of novel phosphorylation sites. 2102 12