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D Koesling

Publications and source records attributed to D Koesling.

At least 55 records · Page 3Linked to original sources
PubMed1996

Characterization of 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one as a heme-site inhibitor of nitric oxide-sensitive guanylyl cyclase.

Nitric oxide (NO) binds with high affinity to the heme of soluble guanylyl cyclase (sGC), resulting in accumulation of the second messenger cGMP in many biological systems. 1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) was recently described as potent and selective inhibitor of sGC, providing an invaluable tool with which to settle the function of the cGMP pathway in NO-mediated signal transduction [Mol. Pharmacol. 48:184-188 (1995)]. The present study investigated the mechanism of ODQ-induced inhibition of purified bovine lung sGC. The drug induced a rightward shift of the concentration-response curves recorded with two different NO donors and a reduction of maximal sGC activity, pointing to a mixed type of inhibition. The time course of NO-stimulated sGC activity determined in the presence of 0.3 microM ODQ showed that the inhibitory effect was time-dependent (half-time approximately 3 min) and virtually complete after about 10 min. The cyclase did not recover from ODQ-induced inhibition upon extensive dilution, pointing to an apparently irreversible inactivation of the enzyme by the quinoxalin. Light absorbance spectroscopy showed that ODQ (0.3 mM) induced a shift of the Soret band of the heme from 431 nm to 393 nm, indicating that ODQ oxidizes the ferrous form of the enzyme to the ferric species, which is though to exhibit only poor NO sensitivity. Together, our results suggest that inhibition of sGC by ODQ is NO-competitive and results in an apparently irreversible oxidation of the prosthetic heme group.

Animals↗
PubMed1995

Functional domains of soluble guanylyl cyclase.

Soluble guanylyl cyclase is a heterodimer consisting of an alpha and beta subunit and stimulation occurs upon binding of NO to a prosthetic group. Little is known about the localization of catalytic and regulatory domains within the subunits of soluble guanylyl cyclase. We used deletion mutagenesis to identify the regions of alpha 1 and beta 1 subunits that are responsible for cGMP production or NO-heme-mediated activation. The amino terminus of the beta 1 subunit was necessary for NO stimulation since deletion of the 64 NH2-terminal amino acids resulted in a mutant with intact basal activity but complete loss of NO activation. The amino terminus of the alpha 1 subunit also appeared to be essential for NO sensitivity since deletion of 131 NH2-terminal amino acids of alpha 1 led to markedly reduced NO activation. These results suggest that NH2-terminal regions of alpha 1 and beta 1 are involved in NO-heme-mediated signal transduction. The NH2 terminally truncated beta 1 subunit exerted a dominant negative effect exclusively on the NO-stimulated activity of the wild type enzyme, further underlining that the regulatory domain is located within the NH2 terminus of the enzyme. Aside for the structural implications, the mutant represents a powerful tool to investigate nitric oxide-sensitive signaling pathways. Coexpression of the COOH-terminal halves of alpha 1 and beta 1 were sufficient for basal cGMP production while either of the halves expressed alone was inactive. Therefore the COOH-terminal regions appear to contain sufficient information for dimerization and basal enzymatic activity. Thus, we provide the first evidence that the regulatory and catalytic properties of soluble guanylyl cyclase can be attributed to different regions of the subunits and that the catalytic domain can be functionally expressed separately from the NH2-terminal regulatory domain. Taken together with findings on the membrane bound enzyme form, guanylyl cyclases, appear to resemble fusion proteins where different regulatory domains have been joined with a common cGMP-forming segment.

Animals↗
PubMed1995

A variant of the alpha 2 subunit of soluble guanylyl cyclase contains an insert homologous to a region within adenylyl cyclases and functions as a dominant negative protein.

A variant of the alpha 2 subunit of soluble guanylyl cyclase (alpha 2i) containing 31 additional amino acids was identified in a number of cell lines and tissues. The in-frame sequence of the insert was within the proposed catalytic domain of guanylyl cyclases and was homologous to a region within the putative catalytic domain of adenylyl cyclases. Messenger RNA for the new variant was detected in some but not all cell lines and tissues expressing the alpha 2 subunit. The novel form, as well as the alpha 2 subunit lacking the insert, were coexpressed with the beta 1 subunit in Sf9 and COS-7 cells; alpha 2/beta 1 coexpression yielded a NO-sensitive recombinant protein, whereas the coexpressed alpha 2i/beta 1 subunits exhibited no guanylyl or adenylyl cyclase activities. Because both subunits (alpha 2i/beta 1) copurified, the novel variant retains its ability to heterodimerize. In coexpression experiments, the alpha 2i subunit competed with the alpha 2 subunit for dimerization with the beta 1 subunit, thereby reducing alpha 2/beta 1-catalyzed guanylyl cyclase activity. These data show that the novel variant functions as a dominant negative protein and that post-transcriptional mRNA processing represents a potential mechanism for regulation of NO-sensitive guanylyl cyclase activity.

Adenylyl Cyclases↗
PubMed1995

Peroxynitrite-induced accumulation of cyclic GMP in endothelial cells and stimulation of purified soluble guanylyl cyclase. Dependence on glutathione and possible role of S-nitrosation.

Peroxynitrite (ONOO-) is widely recognized as mediator of NO toxicity, but recent studies have indicated that this compound may also have physiological activity and induce vascular relaxation as well as inhibition of platelet aggregation. We found that ONOO- induced a pronounced increase in endothelial cyclic GMP levels, and that this effect was significantly attenuated by pretreatment of the cells with GSH-depleting agents. In the presence of 2 mM GSH, ONOO- stimulated purified soluble guanylyl cyclase with a half-maximally effective concentration of about 20 microM. In contrast to the NO donor 2,2-Diethyl-1-nitroso-oxyhydrazine sodium salt (DEA/NO), ONOO- was completely inactive in the absence of GSH, indicating that thiol-mediated bioactivation of ONOO- is involved in enzyme stimulation. Studies on the reaction between ONOO- and GSH revealed that about 1% of ONOO- was non-enzymatically converted to S-nitrosoglutathione. The authentic nitrosothiol was found to be stable in solution, but slowly decomposed in the presence of GSH. GSH-induced decomposition of S-nitrosoglutathione was apparently catalyzed by trace metals and was accompanied by a sustained release of NO and a 40-100-fold increase in its potency to stimulate purified soluble guanylyl cyclase. Our data suggest that the biologic activity of ONOO- involves S-nitrosation of cellular thiols resulting in NO-mediated cyclic GMP accumulation.

Animals↗
PubMed1995

Basis of guanylate cyclase activation by carbon monoxide.

Kinetics of CO association with guanylate cyclase [GTP pyrophosphate-lyase (cyclizing), EC 4.6.1.2] and dissociation from carboxy guanylate cyclase have been studied at pH 7.5 by flash photolysis, yielding rate constants at 23 degrees C of 1.2 +/- 0.1 x 10(5) M-1.sec-1 and 28 +/- 2 sec-1, respectively. While the CO combination rate constant is the same as for the T state of hemoglobin, the CO dissociation rate constant is much higher than expected for a six-coordinate carboxyheme protein; yet the absorption spectrum is indicative of a six-coordinate heme. The two observations are reconciled by a reaction mechanism in which CO dissociation proceeds via a five-coordinate intermediate. This intermediate is structurally very similar to the five-coordinate nitrosyl heme derivative of guanylate cyclase and is presumably responsible for the observed 4-fold activation of guanylate cyclase by CO. Thus, we provide a model that explains enzyme activities of the nitrosyl and carboxy forms of the enzyme on the basis of a common mechanism.

Animals↗
PubMed1995

Immunolocalization of soluble guanylyl cyclase subunits in rat kidney.

Stimulation of soluble guanylyl cyclase (SGC) by nitric oxide (NO) results in the generation of cyclic guanosine monophosphate (cGMP). We recently described expression of abundant nitric oxide synthase, the enzyme by which NO is generated from L-arginine in macula densa cells of rat kidney at the protein and mRNA level. In the present study we looked for possible targets of NO in the kidney. By light and electron microscopy, we applied polyclonal antisera against four subunits (alpha 1, alpha 2, beta 1, beta 2) of SGC in immunocytochemical studies of frozen sections of rat kidney. We demonstrate the presence of alpha 1-subunit in glomerular podocytes and of beta 2-subunit in principal cells of the collecting duct. In both cell types a cytosolic localization was evident from ultrastructural analysis. Regarding the collecting duct, NO was shown by other authors to inhibit sodium reabsorption in cultured mouse cortical collecting duct principal cells. In podocytes NO may relax the contractile system of podocyte food processes, the tone of which has been suggested to counteract the elastic distension of the capillary wall.

Animals↗
PubMed1994

Mutation of His-105 in the beta 1 subunit yields a nitric oxide-insensitive form of soluble guanylyl cyclase.

Soluble guanylyl cyclase [GTP pyrophosphate-lyase (cyclizing); EC 4.6.1.2] is a hemoprotein that exists as a heterodimer; the heme moiety has been proposed to bind nitric oxide, resulting in a dramatic activation of the enzyme. Mutation of six conserved His residues reduced but did not abolish nitric oxide stimulation whereas a change of His-105 to Phe in the beta 1 subunit yielded a heterodimer that retained basal cyclase activity but failed to respond to nitric oxide. Heme was not detected as a component of the mutant heterodimer and protophorphyrin IX failed to stimulate enzyme activity. The activity of the His mutant was almost identical to that of the wild-type enzyme in the presence of KCN, suggesting that disruption of heme binding is the principal effect of the mutation. Thus, the mutation provides a means to inhibit the nitric oxide-sensitive guanylyl cyclase signaling pathway.

Amino Acid Sequence↗
PubMed1992

Characterization of soluble platelet guanylyl cyclase with peptide antibodies.

Soluble guanylyl cyclase partially purified from bovine and human platelets was characterized with antibodies raised against synthetic peptides corresponding to different sequences of the alpha 1- and beta 1-subunits of the bovine lung enzyme. On immunoblots, the platelet guanylyl cyclase was recognized by the four antisera used, with the exception of an antiserum against the C-terminus of the beta 1-subunit which did not react with the human platelet but with the bovine platelet beta 1-subunit. Furthermore the human platelet beta 1-subunit exhibited a slightly lower molecular mass than the bovine protein. The C-terminal antibodies precipitated native platelet and lung guanylyl cyclase activity. In contrast an antibody against a peptide out of the putative catalytic domain, which is highly conserved between all guanylyl cyclases sequenced so far, did not precipitate native guanylyl cyclase, although it recognized both subunits on immunoblots, suggesting that the respective amino acid sequence is located in an inner site of the protein.

Amino Acid Sequence↗
PubMed1991

Molecular cloning and expression of a new alpha-subunit of soluble guanylyl cyclase. Interchangeability of the alpha-subunits of the enzyme.

A cDNA coding for a new subunit of soluble guanylyl cyclase with a calculated molecular mass of 81.7 kDa was cloned and sequenced. On the basis of sequence homology, the new subunit appears to be an isoform of the alpha 1-subunit and was designated alpha 2 as the new subunit is very similar to the alpha 1-subunit in the middle and C-terminal part; it is quite diverse in the N-terminal part. Preceding experiments had shown that coexpression of the alpha 1- and beta 1-subunits is necessary to obtain a catalytically active guanylyl cyclase in COS cells [(1990) FEBS Lett. 272, 221-223]. The finding that the alpha 2-subunit was able to replace the alpha 1- but not the beta 1-subunit in expression experiments demonstrates the interchangeability of the alpha-subunit isoforms of soluble guanylyl cyclase.

Amino Acid Sequence↗
PubMed1991

Sequence homologies between guanylyl cyclases and structural analogies to other signal-transducing proteins.

The cyclic GMP-forming enzyme guanylyl cyclase exists in cytosolic and in membrane-bound forms differing in structure and regulations. Determination of the primary structures of the guanylyl cyclases revealed that the cytosolic enzyme form consists of two similar subunits and that membrane-bound guanylyl cyclases represent enzyme forms in which the catalytic part is located in an intracellular, C-terminal domain and is regulated by an extracellular, N-terminal receptor domain. A domain of 250 amino acids conserved in all guanylyl cyclases appears to be required for the formation of cyclic nucleotide, as this homologous domain is also found in the cytosolic regions of the adenylyl cyclase. The general structures of guanylyl cyclases shows similarities with other signal transducing enzymes such as protein-tyrosine phosphatases and protein-tyrosine kinases, which also exist in cytosolic and receptor-linked forms.

Adenylyl Cyclases↗
PubMed1991

Guanylyl cyclases, a growing family of signal-transducing enzymes.

Guanylyl cyclases, which catalyze the formation of the intracellular signal molecule cyclic GMP from GTP, display structural features similar to other signal-transducing enzymes such as protein tyrosine-kinases and protein tyrosine-phosphatases. So far, three isoforms of mammalian membrane-bound guanylyl cyclases (GC-A, GC-B, GC-C), which are stimulated by either natriuretic peptides (GC-A, GC-B) or by the enterotoxin of Escherichia coli (GC-C), have been identified. These proteins belong to the group of receptor-linked enzymes, with different NH2-terminal extracellular receptor domains coupled to a common intracellular catalytic domain. In contrast to the membrane-bound enzymes, the heme-containing soluble guanylyl cyclase is stimulated by NO and NO-containing compounds and consists of two subunits (alpha 1 and beta 1). Both subunits contain the putative catalytic domain, which is conserved in the membrane-bound guanylyl cyclases and is found twice in adenylyl cyclases. Coexpression of the alpha 1- and beta 1-subunit is required to yield a catalytically active enzyme. Recently, another subunit of soluble guanylyl cyclase was identified and designated beta 2, revealing heterogeneity among the subunits of soluble guanylyl cyclase. Thus, different enzyme subunits may be expressed in a tissue-specific manner, leading to the assembly of various heterodimeric enzyme forms. The implications concerning the physiological regulation of soluble guanylyl cyclase are not known, but different mechanisms of soluble enzyme activation may be due to heterogeneity among the subunits of soluble guanylyl cyclase.

Amino Acid Sequence↗
PubMed1990

Expression of soluble guanylyl cyclase. Catalytic activity requires two enzyme subunits.

Purified soluble guanylyl cyclase consists of two subunits (70 and 73 kDa) whose primary structures were recently determined. The availability of cDNA clones coding for either subunit allowed to study the question of the functional roles of the two subunits in expression experiments. Enzyme subunits were expressed in COS-7 cells by transfection with expression vectors containing the coding region for the 70 of 73 kDa subunit of the enzyme. No significant elevation in the activity of soluble guanylyl cyclase was observed in cells transfected with cDNA coding for one of the subunits. In contrast, transfection of cells with cDNAs coding for both subunits resulted in a marked increase in activity of soluble guanylyl cyclase. Enzyme activity was stimulated about 50-fold by sodium nitroprusside. The results indicate that formation of cyclic GMP by soluble guanylyl cyclase requires both 70 and 73 kDa subunits.

Animals↗
PubMed1990

Purification of soluble guanylyl cyclase from bovine lung by a new immunoaffinity chromatographic method.

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.

Animals↗
PubMed1990

The primary structure of the larger subunit of soluble guanylyl cyclase from bovine lung. Homology between the two subunits of the enzyme.

The primary structure of the larger subunit of the soluble guanylyl cyclase from bovine lung, which catalyzes the formation of cyclic GMP from GTP, has been determined. Two clones, isolated from two bovine libraries yielded a total of 3261 bp with a coding region of 2073 bp. The open reading frame encodes a protein of 691 amino acids and a molecular mass of 77,500. The deduced amino acid sequence reveals regions which are, to a large extent, homologous to the sequence of the smaller subunit of the enzyme as well as to the sequences of other gyanylyl and adenylyl to a large extent, homologous to the sequence of the smaller subunit of the enzyme as well as to the sequences of other gyanylyl and adenylyl cyclases.

Amino Acid Sequence↗
PubMed1989

G-protein alpha-subunits in cytosolic and membranous fractions of human neutrophils.

In plasma membranes of human neutrophils, we identified two major pertussis toxin substrates of 40 kDa Mr with pI values of 5.30 and 5.37. Only the acidic of the two substrates was also present in neutrophil cytosol. Two-dimensional tryptic peptide maps revealed a high degree of homology of cytosolic and particulate substrates. Purified G-protein beta gamma-complex stimulated pertussis toxin-catalyzed [32P]ADP-ribosylation of membranous and cytosolic substrates of neutrophils less than 2-fold and 6-fold, respectively. Hydrodynamic properties of the cytosolic substrate strongly suggested that it exists as a monomer. Purified G-protein beta gamma-complex increased the s20,w value of the cytosolic substrate from 3.3 S to 4.0 S. The GTP analogue, guanosine 5'-O-(3-thiotriphosphate), promoted the release of pertussis toxin substrates from plasma membranes. An antiserum raised against a sequence specific for the Gi2 alpha-subunit reacted with 39-40 kDa proteins in plasma membranes and with an apparently single 40 kDa protein in cytosol. We conclude that neutrophil cytosol contains monomeric Gi2 alpha-subunits which--by interacting with hydrophobic beta gamma-complexes--may reversibly bind to the plasma membrane.

Cell Membrane↗