Rate, extent and concentration dependence of histamine-evoked Weibel-Palade body exocytosis determined from individual fusion events in human endothelial cells.

The rate, concentration dependence and extent of histamine-evoked Weibel-Palade body (WPB) exocytosis were investigated with time-resolved fluorescence microscopy in cultured human umbilical vein endothelial cells expressing WPB-targeted chimeras of enhanced green fluorescent protein (EGFP). Exocytosis of single WPBs was characterized by an increase in EGFP fluorescence, morphological changes and release of WPB contents. The fluorescence increase was due to a rise of intra-WPB pH from resting levels, estimated as pH 5.45+/-0.26 (s.d., n=144), to pH 7.40. It coincided with uptake of extracellular Alexa-647, indicating the formation of a fusion pore, prior to loss of fluorescent contents. Delays between the increase in intracellular free calcium ion concentration evoked by histamine and the first fusion event were 10.0+/-4.42 s (n=9 cells) at 0.3 microM histamine and 1.57+/-0.21 s (n=15 cells) at 100 microM histamine, indicating the existence of a slow process or processes in histamine-evoked WPB exocytosis. The maximum rates of exocytosis were 1.20+/-0.16 WPB s(-1) (n=9) at 0.3 microM and 3.66+/-0.45 WPB s(-1) at 100 microM histamine (n=15). These occurred 2-5 s after histamine addition and declined to lower rates with continued stimulation. The initial delays and maximal rate of exocytosis were unaffected by removal of external Ca2+ indicating that the initial burst of secretion is driven by Ca2+ release from internal stores, but sustained exocytosis required external Ca2+. Data were compared to exocytosis evoked by a maximal concentration of the strong secretagogue ionomycin (1 microM), for which there was a delay between calcium elevation and secretion of 1.67+/-0.24 s (n=6), and a peak fusion rate of approximately 10 WPB s(-1).

Structural and catalytic properties and homology modelling of the human nucleoside diphosphate kinase C, product of the DRnm23 gene.

The human DRnm23 gene was identified by differential screening of a cDNA library obtained from chronic myeloid leukaemia-blast crisis primary cells. The over-expression of this gene inhibits differentiation and induces the apoptosis of myeloid precursor cell lines. We overproduced in bacteria a truncated form of the encoded protein lacking the first 17 N-terminal amino acids. This truncated protein was called nucleoside diphosphate (NDP) kinase CDelta. NDP kinase CDelta had similar kinetic properties to the major human NDP kinases A and B, but was significantly more stable to denaturation by urea and heat. Analysis of denaturation by urea, using size exclusion chromatography, indicated unfolding without the dissociation of subunits, whereas renaturation occurred via a folded monomer. The stability of the protein depended primarily on subunit interactions. Homology modelling of the structure of NDP kinase CDelta, based on the crystal structure of NDP kinase B, indicated that NDP kinase CDelta had several additional stabilizing interactions. The overall structure of the two enzymes appears to be identical because NDP kinase CDelta readily formed mixed hexamers with NDP kinase A. It is possible that mixed hexamers can be observed in vivo.

Quaternary structure of nucleoside diphosphate kinases.

Nucleoside (NDP) diphosphate kinases are oligomeric enzymes. Most are hexameric, but some bacterial enzymes are tetrameric. Hexamers and tetramers are constructed by assembling identical dimers. The hexameric structure is important for protein stability, as demonstrated by studies with natural mutants (the Killer-of-prune mutant of Drosophila NDP kinase and the S120G mutant of the human NDP kinase A in neuroblastomas) and with mutants obtained by site-directed mutagenesis. It is also essential for enzymic activity. The function of the tetrameric structure is unclear.

Association of calmodulin with cytoskeletal structures at different stages of HeLa cell division, visualized by a calmodulin-EGFP fusion protein.

The fusion protein of calmodulin (CaM) with the enhanced green fluorescent protein EGFP has been expressed in a stably transfected HeLa cell line in order to visualise the localisation of calmodulin during the cell cycle on a continuous basis in live cells, and for immunofluorescence colocalisation with cytoskeletal structures. High-resolution images of CaM-EGFP in the mitotic apparatus show the characteristic strongly convoluted structure of the centrosome. CaM-EGFP also apparently associates with both polar and mitotic microtubules, and with a specific intracentrosomal structure. During cytokinesis, CaM-EGFP is also found decorating selected oriented filaments in close proximity to microtubules in the midbody region. In interphase cells, it is seen with filamentous and punctuate localisation at the nuclear envelope. The intensity and continuity of the CaM-EGFP images suggest that a significant fraction of the cellular calmodulin remains attached to cytoplasmic structures during the cell cycle.

nm23-H4, a new member of the family of human nm23/nucleoside diphosphate kinase genes localised on chromosome 16p13.

A novel human nm23/nucleoside diphosphate (NDP) kinase gene, called nm23-H4, was identified by screening a human stomach cDNA library with a probe generated by amplification by reverse transcription-polymerase chain reaction. The primers were designed from publicly available database cDNA sequences selected according to their homology to the human nn23-H1 putative metastasis suppressor gene. The full-length cDNA sequence predicts a 187 amino acid protein possessing the region homologous to NDP kinases with all residues crucial for nucleotide binding and catalysis, strongly suggesting that Nm23-H4 possesses NDP kinase activity. It shares 56, 55 and 60% identity with Nm23-H1, Nm23-H2 and DR-Nm23, respectively, the other human Nm23 proteins isolated so far. Compared with these proteins, Nm23-H4 contains an additional NH2-terminal region that is rich in positively charged residues and could indicate routing to mitochondria. The nm23-H4 gene has been localised to human chromosomal band 16p13.3. The corresponding 1.2 kb mRNA is widely distributed and expressed in a tissue-dependent manner, being found at very high levels in prostate, heart, liver, small intestine and skeletal muscle tissues and in low amounts in the brain and in blood leucocytes. Nm23-H4 naturally possesses the Pro-Ser substitution equivalent to the K-pn mutation (P97S) of Drosophila.

Thermal stability of hexameric and tetrameric nucleoside diphosphate kinases. Effect of subunit interaction.

The eukaryotic nucleoside diphosphate (NDP) kinases are hexamers, while the bacterial NDP kinases are tetramers made of small, single domain subunits. These enzymes represent an ideal model for studying the effect of subunit interaction on protein stability. The thermostability of NDP kinases of each class was studied by differential scanning calorimetry and biochemical methods. The hexameric NDP kinase from Dictyostelium discoideum displays one single, irreversible differential scanning calorimetry peak (Tm 62 degrees C) over a broad protein concentration, indicating a single step denaturation. The thermal stability of the protein was increased by ADP. The P105G substitution, which affects a loop implicated in subunit contacts, yields a protein that reversibly dissociates to folded monomers at 38 degrees C before the irreversible denaturation occurs (Tm 47 degrees C). ADP delays the dissociation, but does not change the Tm. These data indicate a "coupling" of the quaternary structure with the tertiary structure in the wild-type, but not in the mutated protein. We describe the x-ray structure of the P105G mutant at 2.2-A resolution. It is very similar to that of the wild-type protein. Therefore, a minimal change in the structure leads to a dramatic change of protein thermostability. The NDP kinase from Escherichia coli behaves like the P105G mutant of the Dictyostelium NDP kinase. The detailed study of their thermostability is important, since biological effects of thermolabile NDP kinases have been described in several organisms.