Summary: Coronary artery ectasia (CAE) is found in 0.3-5% of patients undergoing coronary angiography. Atherosclerosis is the main cause, followed by Kawasaki disease and infectious emboli. The exact pathogenesis has not been diagnosed as yet, but an inflammatory process is underlying. Symptoms, if present, are usually related to myocardial ischemia. Angiography is the mainstay for diagnosis. The prognosis is generally favorable. Thromboembolic complications are rare with antiplatelet therapy, and spontaneous rupture generally is rare but occurs more commonly in Kawasaki disease. Management varies from antithrombotic therapy to surgical ligation. Controlling coronary heart disease risk factors sharply affects the prognosis in patients with CAE.

Soluble guanylyl cyclase was purified from bovine lung by an immunoaffinity chromatographic method using IgG fractions of antisera against a synthetic peptide of the C-terminus of the 70-kDa subunit of the enzyme. After anion-exchange chromatography, the enzyme was bound to an immunoaffinity column and was eluted with the synthetic peptide. This method allowed the convenient isolation of 2 mg of apparently homogeneous enzyme from 40 g cytosolic proteins. The enzyme had an apparent molecular mass of about 150 kDa and consisted of two subunits (70 kDa and 73 kDa) as determined by gel permeation fast protein liquid chromatography and SDS/PAGE. The basal activities determined in the presence of Mg2+ and Mn2+ were 10-20 nmol.min-1.mg-1 and 80-100 nmol.min-1.mg-1, respectively. The enzyme exhibited an ultraviolet-visible absorption spectrum typical for hemoproteins, with a Soret band at 430 nm. The purified enzyme was stimulated by NO-containing compounds. Maximal enzyme activities measured in the presence of sodium nitroprusside were 1.2-2.4 mumol.min-1.mg-1 (half-maximal effect of sodium nitroprusside at 1.3-1.9 microM) and 0.9-1.8 mumol.min-1.mg-1 (half-maximal effect at 0.28-0.41 microM sodium nitroprusside) in the presence of Mg2+ and Mn2+, respectively. The method developed for the large-scale purification of soluble guanylyl cyclase by immunoaffinity chromatography, using synthetic peptides for the elution of the enzyme, appears to be superior to previously described methods. As antibodies against synthetic peptides corresponding to deduced amino acid sequences of the respective protein are easily obtained, the described method may be suitable for a convenient large-scale purification of various proteins.

G protein ␤␥ dimers can be phosphorylated in membranes from various tissues by GTP at a histidine residue in the ␤ subunit. The phosphate is high energetic and can be transferred onto GDP leading to formation of GTP. Purified G␤␥ dimers do not display autophosphorylation, indicating the involvement of a separate protein kinase. We therefore enriched the G␤-phosphorylating activity present in preparations of the retinal G protein transducin and in partially purified G i/o proteins from bovine brain. Immunoblots, autophosphorylation, and enzymatic activity measurements demonstrated enriched nucleoside diphosphate kinase (NDPK) B in both preparations, together with residual G␤␥ dimers. In the retinal NDPK B-enriched fractions, a G␤-specific antiserum co-precipitated phosphorylated NDPK B, and an antiserum against the human NDPK co-precipitated phosphorylated G␤␥. In addition, the NDPK-containing fractions from bovine brain reconstituted the phosphorylation of purified G␤␥. For identification of the phosphorylated histidine residue, bovine brain G␤␥ and G t ␤␥ were thiophosphorylated with guanosine 5 -O-(3-[ 35 S]thio)triphosphate, followed by digestion with endoproteinase Glu-C and trypsin, separation of the resulting peptides by gel electrophoresis and high pressure liquid chromatography, respectively, and sequencing of the radioactive peptides. The sequence information produced by both methods identified specific labeled fragments of bovine G␤ 1 that overlapped in the heptapeptide, Leu-Met-Thr-Tyr-Ser-His-Asp (amino acids 261-267). We conclude that NDPK B forms complexes with G␤␥ dimers and contributes to G protein activation by increasing the high energetic phosphate transfer onto GDP via intermediately phosphorylated His-266 in G␤ 1 subunits.Heterotrimeric G proteins play a pivotal role in many signal transduction pathways in eukaryotic cells. They consist of a guanine nucleotide-binding ␣ subunit (40 -52 kDa), a ␤ subunit (33-43 kDa), and a ␥ subunit (6 -10 kDa). The latter two act as a functional unit and only dissociate upon denaturation. Both G␣ and G␤␥ are required for receptor-induced G protein activation and can trigger effector functions (for reviews see Refs. 1-3). Heterotrimeric G proteins are activated by a GDP/GTP exchange catalyzed by G protein-coupled receptors. Furthermore, we and other laboratories provided evidence that phosphotransfer reactions can participate in G protein activation in vitro by formation of GTP. There is amble evidence that nucleoside diphosphate kinase (NDPK) 1 contributes to G protein activation by replenishment of GTP from ATP and GDP (for reviews see Refs. 4 -6). Hypotheses suggesting a direct in situ phosphorylation of GDP bound to G␣ and monomeric G proteins (7-9) are most likely based on artifacts. Also complex formation of NDPK with G proteins and channeling of NDPKformed GTP into G␣ (10, 11) have not yet been proven beyond doubt.A phosphotransfer reaction that uses G␤ subunits as phosphorylated intermediates has been observed in various tissues (12)(13)(14)(15)(16). ...

Formation of GTP by nucleoside diphosphate kinase (NDPK) can contribute to G protein activation in vitro.To study the effect of NDPK on G protein activity in living cells, the NDPK isoforms A and B were stably expressed in H10 cells, a cell line derived from neonatal rat cardiomyocytes. Overexpression of either NDPK isoform had no effect on cellular GTP and ATP levels, basal cAMP levels, basal adenylyl cyclase activity, and the expression of G s ␣ and G i ␣ proteins. However, co-expression of G s ␣ led to an increase in cAMP synthesis that was largely enhanced by the expression of NDPK B, but not NDPK A, and that was confirmed by direct measurement of adenylyl cyclase activity. Cells expressing an inactive NDPK B mutant (H118N) exhibited a decreased cAMP formation in response to G s ␣. Co-immunoprecipitation studies demonstrated a complex formation of the NDPK with G␤␥ dimers. The overexpression of NDPK B, but not its inactive mutant or NDPK A, increased the phosphorylation of G␤ subunits. In summary, our data demonstrate a specific NDPK B-mediated activation of a G protein in intact cells, which is apparently caused by formation of NDPK B⅐G␤␥ complexes and which appears to contribute to the receptor-independent activation of heterotrimeric G proteins. Nucleoside diphosphate kinase (NDPK)1 catalyzes the transfer of terminal phosphate groups from 5Ј-triphosphate to 5Ј-diphosphate nucleotides. In the cell, the major reaction is the phosphate transfer from ATP to other NDPs to maintain the levels of NTPs, especially the relatively high level of GTP. Only a small fraction of cellular NDPK binds to the plasma membrane, where it may serve the synthesis of GTP, required for the activation of G proteins (1-3). An activation of G proteins by NDPK has been disputed for more than 10 years. Although numerous in vitro studies (4 -7) have shown G protein activation through the enzymatic activity of NDPK (synthesis of GTP from a nucleoside triphosphate and GDP), the specificity of this phenomenon has been questioned (8, 9). Particularly in the intact cell, where GTP concentrations are in the upper micromolar range, evidence for a mechanism beyond the sole synthesis of GTP appears mandatory to support this hypothesis. On the other hand, we have shown recently (10) that NDPK activates G proteins and regulates adenylyl cyclase activity in canine cardiac sarcolemmal membranes. This activation required the catalytic activity of NDPK (synthesis of GTP) but was clearly distinct from the effect of exogenous GTP, suggesting a more direct interaction of NDPK and G proteins.Evaluation of direct G protein activation through phosphotransfer by NDPK is associated with substantial methodological constraints. Mainly, GDP is released spontaneously from G proteins and may then serve as a free substrate for phosphorylation by the NDPK (8). Approaches to immobilize the bound GDP at the G protein (11) are associated with protein denaturation, which in turn may lead to unspecific protein phosphorylation by the NDPK (12). In addition, structural con...

We conclude that plasma endothelin-1 is increased at high altitude, but whether or not it represents an important pathogenetic factor for pulmonary hypertension remains to be investigated.