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Biomedical subjects

G Schultz

Publications and source records attributed to G Schultz.

At least 271 records · Page 15Linked to original sources
PubMed1985

Localization, purification, and characterization of shikimate oxidoreductase-dehydroquinate hydrolyase from stroma of spinach chloroplasts.

The stroma of chloroplasts is probably the sole site of the shikimate pathway enzymes shikimate oxidoreductase/dehydroquinate hydrolyase (SORase/DHQase) in spinach leaves. (a) The chromatographic behavior of the bifunctional protein SORase/DHQase on several separation materials with extracts from stroma compared with leaf extracts showed only one peak of enzymic activity originating from the stroma. (b) Polyacrylamide gel electrophoresis (PAGE) of these extracts followed by specific staining resulted in the same pattern without a band of extraplastidic enzyme. (c) In protoplast fractionation experiments it was shown that SORase/DHQase was present only in the soluble chloroplast protein fraction.An improved purification procedure for SORase/DHQase from stroma of chloroplasts, yield 40%, 1600 times as pure, gave essentially one protein band on sodium dodecyl sulfate-PAGE. Our results demonstrate that both enzyme functions are carried out by a single polypeptide. Nondenaturing PAGE exhibited a pattern of four bands with SORase/DHQase showing that they differ in charge but not in their molecular weight. Molecular weight was determined to be 67 kilodaltons (gel filtration) and 59 kilodaltons (PAGE) for all four forms. It was proven they were not due to artifacts. The four forms show similar kinetic properties, their K(m) and pH optima differing only very slightly. Response to some metabolites is reported.

Journal Article↗
PubMed1985

[Principles of transmembranous signal transduction in the action of hormones and neurotransmitters].

In the membranous signal transduction process, hormone-binding to receptors causes receptor interaction with signal-transducing components; these components transfer the stimulus to effector systems, which generate intracellular signals. Several guanine nucleotide-binding proteins (N- or G-proteins) have been identified as membranous signal-transducing components. Two N-proteins are involved in the hormonal regulation of adenylate cyclase activity, one of which being stimulatory (Ns), the other one being inhibitory (Ni). Ns, Ni and a third N-protein, No, whose function is unknown, occur ubiquitously. On the other hand, transducin, an N-protein, which functionally couples light-activated rhodopsin to a cGMP phosphodiesterase, is specific for the retina. In addition to their established role as transducers regulating adenylate cyclase and retinal cGMP phosphodiesterase, N-proteins proteins may be involved in two mechanisms by which the cytoplasmic calcium concentration is elevated, i.e. hormonal stimulation of a phospholipase C catalyzing phosphatidyl-inositol 4,5-diphosphate hydrolysis (Pi response) and hormone-induced opening of receptor-operated calcium channels; the membrane-bound forms of cAMP phosphodiesterase and guanylate cyclase, stimulated by insulin and atrial natriuretic factor, respectively, are also likely to be regulated via N-proteins. Guanine nucleotide-binding proteins appear to play a universal role in transmembranous signalling processes, controlling effector systems (i.e. enzymes and ion channels) that regulate cytoplasmic concentrations of intracellular messengers such as cyclic AMP, cyclic GMP and calcium.

Adenylyl Cyclases↗
PubMed1984

Mechanism of pertussis toxin action on the adenylate cyclase system. Inhibition of the turn-on reaction of the inhibitory regulatory site.

The activation reaction of the inhibitory guanine nucleotide-binding regulatory site of the adenylate cyclase system was studied in membranes of rat adipocytes, S49 lymphoma wild-type cells and their cyc- variants, pretreated without and with the Bordetella pertussis toxin, islet-activating protein (IAP), by measuring the kinetics of adenylate cyclase inhibition by the stable GTP analogue, guanosine 5'-[gamma-thio]triphosphate (GTP[S]). The IAP treatment, which caused a loss of GTP and hormone-induced adenylate cyclase inhibition, did not prevent enzyme inhibition by the stable GTP analogue. However, in either cell type studied, pretreated with IAP, the lag phase of GTP[S] inhibitory action was largely increased by about fivefold compared to control membranes. Similar to the controls, the lag phase of GTP[S] inhibition of adenylate cyclase in membranes of IAP-pretreated cells was shortened in the presence of an inhibitory hormone. Furthermore, the lag phase of inhibition by GTP[S] was decreased with increasing concentrations of Mg2+. The data indicate that the pertussis toxin does not principally prevent an interaction of the inhibitory guanine nucleotide regulatory site of the adenylate cyclase system with either the catalytic moiety or an inhibitory hormone receptor. The data, furthermore, suggest that the toxin inhibits the activation reaction (turn-on reaction) of the inhibitory coupling component. This inhibition, which may take place at a Mg2+-binding site, can account for the observed functional loss of GTP and hormone-induced adenylate cyclase inhibition after IAP treatment.

Adenylate Cyclase Toxin↗
PubMed1984

Pyruvate-Derived Amino Acids in Spinach Chloroplasts : Synthesis and Regulation during Photosynthetic Carbon Metabolism.

A probable carbon flow from the Calvin cycle to branched chain amino acids and lipids via phosphoenolpyruvate (PEP) and pyruvate was examined in spinach (Spinacia oleracea) chloroplasts. The interpendence of metabolic pathways in and outside chloroplasts as well as product and feedback inhibition were studied. It was shown that alanine, aromatic, and small amounts of branched chain amino acids were formed from bicarbonate in purified intact chloroplasts. Addition of PEP only favored formation of aromatic amino acids. Mechanisms of regulation remained unclear. Concentrations of PEP and pyruvate within the chloroplast impermeable space during photosynthetic carbon fixation were 15 times higher than in the reaction medium. A direct carbon flow to pyruvate was identified (0.1 micromoles per milligram chlorophyll per hour). Pyruvate was taken up by intact chloroplasts slowly, leading to the formation of lysine, alanine, valine, and leucine plus isoleucine (approximate ratios, 100-500:60-100:40-100:2-10). The K(m) for the formation of valine and leucine plus isoleucine was estimated to be 0.1 millimolar. Ten micromolar glutamate optimized the transamination reaction regardless of whether bicarbonate or pyruvate was being applied. Alanine and valine formation was enhanced by the addition of acetate to the reaction mixture. The enhancement probably resulted from an inhibition of pyruvate dehydrogenase by acetyl-S-coenzyme A formed from acetate, and resulting accumulation of hydroxyethylthiamine diphosphate and pyruvate. High concentrations of valine and isoleucine inhibited their own and each others synthesis and enhanced alanine formation. When pyruvate was applied, only amino acids were formed; when complemented with bicarbonate, fatty acids were formed as well. This is probably the result of a requirement of acetyl-S-coenzyme A-carboxylase for bicarbonate.

Journal Article↗
PubMed1983

Inhibition of NS-protein-stimulated human-platelet adenylate cyclase by epinephrine and stable GTP analogs.

The influence of purified, preactivated NS protein, mediating adenylate cyclase stimulation by hormones and guanine nucleotides, was studied on adenylate cyclase activity in membranes of human platelets. The preactivated coupling protein caused a large increase in platelet enzyme activity. The activation occurred after a short lag phase and exhibited saturation. NS protein increased the V of the platelet adenylate cyclase without change in the enzyme's affinity for the substrate, MgATP, whereas the apparent affinity for Mg2+ was increased by more than one order of magnitude. The NS-protein-activated human platelet adenylate cyclase was inhibited by the hormone, epinephrine, and by the stable GTP analogs, guanosine 5'-[beta, gamma-imido]triphosphate and guanosine 5'-[gamma-thio]triphosphate. The stable GTP analog or hormone-induced adenylate cyclase inhibition was not competitive with regard to the concentration of NS protein. Inhibition of NS-protein-stimulated platelet adenylate cyclase caused by stable GTP analogs appeared to be persistent, was amplified by epinephrine and was accompanied by a large decrease in the enzyme's apparent affinity for Mg2+. The data suggest that the hormone-sensitive adenylate cyclase is regulated by two guanine-nucleotide-binding sites, NS and Ni, mediating enzyme stimulation and inhibition, respectively, by hormones and guanine nucleotides, and that the two coupling components interact in a non-competitive manner with the adenylate cyclase, exhibiting high and low affinity for Mg2+ when affected by NS and Ni, respectively.

Adenylyl Cyclase Inhibitors↗
PubMed1983

Adenylate cyclase inhibition and GTPase stimulation by somatostatin in S49 lymphoma cyc- variants are prevented by islet-activating protein.

cyc--Variants of S49 lymphoma cells are defective in the stimulatory guanine nucleotide site of the adenylate cyclase but contain an inhibitory site. Treatment of cyc- cells with islet-activating protein (IAP), which causes ADP-ribosylation of an Mr 40 000 polypeptide in cyc- membranes, abolishes adenylate cyclase inhibition by GTP and the peptide hormone, somatostatin, but not that induced by GTP gamma S. Furthermore, somatostatin-induced stimulation of GTP hydrolysis is lost. Thus, the data indicate that IAP interferes with the adenylate cyclase system by an action at the inhibitory guanine nucleotide site.

Adenylate Cyclase Toxin↗
PubMed1983

Islet-activating protein prevents nicotinic acid-induced GTPase stimulation and GTP but not GTP gamma S-induced adenylate cyclase inhibition in rat adipocytes.

The influence of islet-activating protein (IAP), a Bordetella pertussis toxin, was studied on adenylate cyclase and GTPase activities in rat adipocyte membranes. Pretreatment of rats or intact rat adipocytes with IAP did not affect adenylate cyclase inhibition by the stable GTP analog, GTP gamma S, whereas inhibition by GTP was abolished. Concomitantly, activation of the adipocyte enzyme by sodium and its inhibition by nicotinic acid were prevented. Furthermore, IAP treatment of adipocyte membranes prevented nicotinic acid-induced stimulation of a high affinity GTPase. The data suggest that a GTP-hydrolyzing system involved in the inhibitory regulation of adenylate cyclase is the target of IAP's action.

Adenylate Cyclase Toxin↗
PubMed1983

Activation of human platelet adenylate cyclase by a bovine sperm component.

The interaction of bovine sperm particles and human platelet membranes was studied with regard to adenylate cyclase activity. When sperm and platelet membrane preparations were combined, greater than additive adenylate cyclase activity was measured. Data obtained after inactivation of the sperm enzyme indicated that the observed increase in adenylate cyclase activity was not due to an activation of the sperm cyclase but that a sperm component, which could be extracted from the sperm particles, activated the platelet adenylate cyclase. Platelet cyclase activation by the sperm particles was a saturable and time-dependent process. The extent of activation was particularly high (up to 10-fold) in the presence of a stable GTP analog. Under this condition, the apparent affinity of the platelet enzyme for Mg2+ was increased by about one order of magnitude, whereas with Mn2+ only a minor effect of the sperm particles was observed. Platelet adenylate cyclase stimulation by prostaglandin E1 (up to 20-fold) was more than doubled by the sperm particles, whereas the inhibition of the platelet enzyme by epinephrine was abolished. The sperm factor was trypsin-sensitive and was inactivated by boiling. The data indicate that bovine sperm particles contain an extractable protein, which activates platelet adenylate cyclase probably by inactivating an inhibitory site.

Adenylyl Cyclases↗
PubMed1983

Islet-activating protein impairs alpha 2-adrenoceptor-mediated inhibitory regulation of human platelet adenylate cyclase.

Human platelet adenylate cyclase is stimulated by prostaglandin E1 (PGE1) and inhibited by adrenaline acting via alpha 2-adrenoceptors. Both hormonal factors increase GTP hydrolysis in platelet membranes by stimulation of a high affinity GTPase. The influence of the Bordetella pertussis toxin, islet-activating protein (IAP), was studied on adrenaline and PGE1-induced GTPase stimulation and on adrenaline-induced adenylate cyclase inhibition. Pretreatment of platelet membranes with IAP greatly reduced the adrenaline-induced inhibition of the platelet adenylate cyclase. At similar concentrations of the toxin, stimulation of GTP hydrolysis caused by adrenaline was impaired. In contrast, pretreatment of platelet membranes with IAP had no effect on stimulation of GTP hydrolysis by the adenylate cyclase-stimulating agent, PGE1, both when studied alone or in combination with adrenaline. On the other hand, cholera toxin reduced the PGE1-induced GTPase stimulation but did not affect the adrenaline-stimulated GTP hydrolysis. The data indicate that there are two separate GTP-hydrolyzing systems in human platelet membranes and that cholera toxin and pertussis toxin selectively affect stimulation of GTP hydrolysis by hormones that stimulate and inhibit adenylate cyclase, respectively.

Adenylate Cyclase Toxin↗
PubMed1983

Pertussis toxin decreases opiate receptor binding and adenylate inhibition in a neuroblastoma x glioma hybrid cell line.

Neuroblastoma X glioma hybrid cells NG108-15 were treated with a toxin derived from Bordetella pertussis. As compared to control cells grown in the absence of toxin, the inhibitory effects of opioid agonists upon cAMP formation were dose-dependently impaired by a non-competitive mechanism. Radioligand binding studies revealed that opioid agonist binding was dramatically reduced in toxin-treated membranes when tested in the presence of Na+/Mg++/GMP-PNP. Further, the potencies of guanine nucleotides to decrease opioid agonist binding were differentially modulated. These studies may facilitate our understanding of the mechanisms responsible for acute and chronic opiate effects.

Adenylyl Cyclase Inhibitors↗
PubMed1983

Occurrence of a hormone-sensitive inhibitory coupling component of the adenylate cyclase in S49 lymphoma cyc- variants.

The influence of somatostatin was studied on cyclic AMP levels and adenylate cyclase activity in cyc- variants of S49 lymphoma cells. These cells are deficient in the guanine nucleotide site that mediates hormone-induced adenylate cyclase stimulation, but their cyclase can be stimulated by forskolin. Somatostatin maximally decreased the 30 microM forskolin-stimulated cyclic AMP levels by 35%. Half-maximal suppression occurred at about 0.1 nM somatostatin. Somatostatin (up to 1 microM) had no effect on the 100 microM forskolin-stimulated adenylate cyclase activity in cyc- membrane preparations when guanine nucleotides were not present. In the presence of GTP, however, which by itself caused a small decrease in activity, somatostatin maximally inhibited the enzyme by 20-25%. GTP was half-maximally effective at 0.1 microM, and half-maximal inhibition by somatostatin was observed at 0.1- 1 nM. In the presence of the stable GTP analog guanosine 5'-O-(3-thiotriphosphate) (1 microM), which decreased the stimulated activity by about 40% after a short lag period, somatostatin (1 microM) did not cause a further decrease in final activity but reduced the lag period by about 50%. The data indicate that membranes of cyc- variants contain a regulatory site that mediates both guanine nucleotide and hormone-induced inhibition of the adenylate cyclase and suggest that the mechanisms of activation and inactivation of this inhibitory site are similar to those of the stimulatory component missing in cyc-membranes.

Adenylyl Cyclases↗