Novel Ca2+-binding protein (CAPS) related to UNC-31 required for Ca2+-activated exocytosis.
Journal - The Journal of biological chemistry (UNITED STATES )
Exocytotic secretion in neuroendocrine cells is activated by cytoplasmic Ca2+ increases. Late post-docking events in dense core vesicle exocytosis in permeable PC12 cells require cytosolic factors for sequential ATP-dependent priming and Ca2+-dependent triggering steps. The cytosolic proteins phosphatidylinositol transfer protein and phosphatidylinositol (4)-phosphate 5-kinase, as well as membrane-bound N-ethylmaleimide-sensitive factor, are required for the ATP-dependent priming step. Following priming, the Ca2+-dependent triggering of vesicle fusion requires an additional cytosolic factor, CAPS, which was purified as a 145-kDa protein. To clarify late Ca2+-dependent events in vesicle fusion, the sequence of rat CAPS cDNA was determined and found to encode a novel protein that is the vertebrate homologue of the Caenorhabditis elegans UNC-31 protein shown genetically to be required for neurosecretion. Recombinant CAPS substituted for cytosol in the Ca2+ triggering step in permeable PC12 cells and exhibited moderate affinity (Kd = 270 microM) Ca2+ binding (2 mol Ca2+/mol CAPS dimer), consistent with a role at a Ca2+-regulated step in exocytosis.
|ISSN : ||0021-9258|
|Mesh Heading : ||Adenosine Triphosphate Amino Acid Sequence Animals Caenorhabditis elegans Calcium Calcium-Binding Proteins Carrier Proteins Helminth Proteins Kinetics Molecular Sequence Data Molecular Weight PC12 Cells Phospholipid Transfer Proteins Phosphotransferases (Alcohol Group Acceptor) Rats metabolism genetics metabolism genetics metabolism|
|Mesh Heading Relevant : ||Caenorhabditis elegans Proteins Exocytosis Membrane Proteins metabolism metabolism metabolism|
A nucleoside diphosphate kinase from Paramecium tetraurelia with protein kinase activity.
Journal - The Journal of eukaryotic microbiology (UNITED STATES )
Nucleoside diphosphate kinase (NDP kinase) from Paramecium was purified to homogeneity. The native enzyme was 80 kDa (by gel filtration), with subunits of 18 and 20 kDa. Near the amino terminus, 15 of 20 residues were identical with those in human NDP kinase, and 17 of 20 with the awd gene product from Drosophila. NDP kinase bound alpha-labeled ATP and GTP, and a photoreactive GTP analog labeled both subunits. Purified NDP kinase underwent autophosphorylation on a histidine and a serine residue using either ATP or GTP as a substrate. The enzyme also catalyzed acid-stable phosphorylation of casein and phosvitin. This protein kinase activity is distinct from the histidine phosphorylation that is part of the NDP kinase catalytic cycle. Antiserum against the purified protein from Paramecium cross-reacted with 16- to 20-kDa proteins in most species tested, and with a larger protein (44 kDa) in Paramecium, Xenopus, and two human lines. The multiple forms (20 and 44 kDa) of the NDP kinase in Paramecium and its protein kinase activity, suggest that the protein is more than a housekeeping enzyme; it may have regulatory roles such as those of the NDP kinase-like awd protein of Drosophila and Nm23 protein of humans.
|ISSN : ||1066-5234|
|Mesh Heading : ||Adenosine Triphosphate Amino Acid Sequence Animals Caseins Cell Line Guanosine Triphosphate Hela Cells Humans Molecular Sequence Data Nucleoside-Diphosphate Kinase PC12 Cells Paramecium tetraurelia Phosphorylation Phosvitin Protein Kinases Protozoan Proteins Rabbits Sequence Homology, Amino Acid metabolism metabolism metabolism chemistry isolation & purification metabolism chemistry isolation & purification chemistry isolation & purification|
|Mesh Heading Relevant : ||metabolism enzymology metabolism metabolism|
Protein substrates for cGMP-dependent protein phosphorylation in cilia of wild type and atalanta mutants of Paramecium.
Journal - Cell motility and the cytoskeleton (UNITED STATES )
In the ciliated protozoan Paramecium, swimming direction is regulated by voltage-gated Ca2+ channels in the ciliary membrane. In response to depolarizing stimuli, intraciliary Ca2+ rises, triggering reversal of the ciliary power stroke and backward swimming. One class of Ca(2+)-unresponsive behavioral mutants of Paramecium, atalanta mutants, cannot swim backward even though they have functional Ca2+ channels in their ciliary membrane. Several atalanta mutants were characterized with regard to several Ca(2+)-dependent activities, but no significant difference between wild type and the mutants was detected. However, one allelic group, atalanta A (initially characterized by Hinrichsen and Kung [1984: Genet. Res. Camb. 43:11-20]), showed a helical swimming path of opposite handedness from that of wild-type cells when detergent-permeabilized cells ("models") were reactivated with MgATP. When cGMP-dependent protein kinase purified from wild-type cells was added to atalanta A models, the handedness of the swimming path was reversed. Cyclic GMP stimulated in vitro phosphorylation of several proteins in isolated cilia, and the pattern of phosphoproteins was very similar for wild type and atalanta mutants, with one exception: a protein of 59 kDa was phosphorylated much less in the mutant ata A. When ciliary proteins were separated by gel electrophoresis and then phosphorylated "on blot" by purified cGMP-dependent protein kinase, phosphoprotein patterns were similar in wild type and ata mutants except that a 48 kDa protein (p48) from ata A3 was more heavily phosphorylated. This difference in p48 phosphorylation was also observed with cGMP-dependent protein kinase purified from ata A3 mutant cells.(ABSTRACT TRUNCATED AT 250 WORDS)
|ISSN : ||0886-1544|
|Mesh Heading : ||Animals Calcium Cilia Cyclic GMP Cyclic GMP-Dependent Protein Kinases Mutation Paramecium Phosphorylation Protozoan Proteins Substrate Specificity metabolism metabolism genetics metabolism|
|Mesh Heading Relevant : ||metabolism metabolism metabolism|