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G Schultz

Publications and source records attributed to G Schultz.

At least 253 records · Page 14Linked to original sources
PubMed1988

Cyclic AMP-independent, dual regulation of voltage-dependent Ca2+ currents by LHRH and somatostatin in a pituitary cell line.

Voltage-dependent Ca2+ currents appear to be involved in the actions of hormones that regulate pituitary secretion. In order to investigate modulation of Ca2+ currents by release-inducing and release-inhibiting hormones, we performed whole-cell clamp experiments in the pituitary cell line GH3. The resting potential was approximately -40 mV; spontaneous action potentials were observed in the majority of cells. Superfusion of cells with the stimulatory hormone, LHRH, depolarized the plasma membrane to approximately -10 mV, whereas the inhibitory hormone, somatostatin, caused hyperpolarization to approximately -60 mV; both hormones suppressed spontaneous action potentials. Under voltage clamp conditions, GH3 cells exhibited slowly and fast inactivating Ca2+ currents. LHRH increased whereas somatostatin decreased the slowly inactivating currents; fast inactivating currents were not affected by these hormones. The stimulatory effect of LHRH was not mimicked by intracellularly applied cAMP. In contrast to vasoactive intestinal peptide and forskolin, LHRH did not activate adenylate cyclase in membranes of GH3 cells, but rather appeared to cause inhibition of the enzyme. Hormonal stimulation and inhibition of inward currents were abolished by pretreatment of the cells with pertussis toxin. In membranes of GH3 cells, we identified a pertussis toxin-sensitive G-protein of the Gi-type and Go. We conclude that LHRH and somatostatin modulate voltage-dependent Ca2+ currents via cAMP-independent mechanisms involving pertussis toxin-sensitive G-proteins. The occurrence of both pertussis toxin-sensitive hormonal stimulation and inhibition of voltage-dependent Ca2+ currents in one cell type suggest that these opposite regulations are mediated by distinct G-proteins.

Animals↗
PubMed1988

Assessment of biological activity of synthetic fragments of transforming growth factor-alpha.

Transforming growth factor-alpha (TGF-alpha) is a single chain polypeptide hormone of 50 amino acids that stimulates growth of some human cancer cells via an autocrine mechanism. The domain(s) of TGF-alpha that bind and activate its receptor have not been reported. Hydrophilicity plots of TGF-alpha indicate three discrete sequences that are theoretically exposed on the hormone's surface and thus potentially able to interact with the TGF-alpha receptor. Fragments of TGF-alpha encompassing these hydrophilic domains were prepared by using solid-phase peptide synthesis (SPPS) techniques and purified by use of high performance liquid chromotography (HPLC). Assessment of biological activity of the TGF-alpha fragments indicated that none of the fragments significantly inhibited binding of EGF to the receptor, stimulated DNA synthesis of cells, inhibited EGF-induced DNA synthesis of cells, stimulated growth of cells in soft agar, or induced phosphorylation of the receptor or p35 protein. These results indicate that the receptor binding domain of TGF-alpha is not totally encompassed by any of the separate fragments tested and probably is formed by multiple separate regions of TGF-alpha.

Amino Acid Sequence↗
PubMed1988

Control of voltage-dependent Ca2+ channels by G protein-coupled receptors.

G proteins act as transducers between membrane receptors activated by extracellular signals and enzymatic effectors controlling the concentration of cytosolic signal molecules such as cAMP, cGMP, inositol phosphates and Ca2+. In some instances, the receptor/G protein-induced changes in the concentration of cytosolic signal molecules correlate with activity changes of voltage-dependent Ca2+ channels. Ca2+ channel modulation, in these cases, requires the participation of protein kinases whose activity is stimulated by cytosolic signal molecules. The respective protein kinases phosphorylate Ca2+ channel-forming proteins or unknown regulatory components. More recent findings suggest another membrane-confined mechanism that does not involve cytosolic signal molecules but rather a more direct control of voltage-dependent Ca2+ channels by G proteins. Modulation of Ca2+ channel activity that follows this apparently membrane-confined mechanism has been described to occur in neuronal, cardiac, and endocrine cells. The G protein involved in the hormonal stimulation of Ca2+ channels in endocrine cells may belong to the family of Gi-type G proteins, which are functionally uncoupled from activating receptors by pertussis toxin. The G protein Gs, which is activated by cholera toxin, may stimulate cardiac Ca2+ channels without the involvement of a cAMP-dependent intermediate step. Hormonal inhibition of Ca2+ channels in neuronal and endocrine cells is mediated by a pertussis toxin-sensitive G protein, possibly Go. Whether G proteins act by binding directly to Ca2+ channels or through interaction with as yet undetermined regulatory components of the plasma membrane remains to be clarified.

Animals↗
PubMed1987

Activation of protein kinase C by cis- and trans-octadecadienoic acids in intact human platelets and its potentiation by diacylglycerol.

Octadecadienoic acids (linoleic acid and linolelaidic acid) and the diacylglycerol, 1-oleoyl-2-acetyl-rac-glycerol (OAG) concentration-dependently induced activation of gel-filtered human platelets, i.e. aggregation and phosphorylation of 20 kDa and 47 kDa peptides. In contrast, octadecenoic acids (oleic and elaidic acid) and octadecanoic (stearic) acid were inactive. Octadecadienoic acid-induced platelet activation was suppressed by the protein kinase C inhibitor, polymyxin B, but not by the cyclooxygenase inhibitor, indomethacin. OAG-induced activation was potentiated by octadecadienoic acids present at non-stimulatory concentrations. Our data suggest that octadecadienoic acids and diacylglycerol synergistically induce platelet activation via protein kinase C. Furthermore, linolelaidic acid may provide a useful experimental tool to study fatty acid regulation of protein kinase C in intact cells.

Blood Platelets↗
PubMed1987

Resolution of transducin subunits by chromatography on blue sepharose.

The retinal guanine nucleotide-binding protein, transducin (TD), was subjected to chromatography on Blue Sepharose (BLS). A simple two-step protocol was developed, allowing the resolution of the alpha-subunit and the beta gamma-complex of the protein extracted from bovine retina by the use of a poorly hydrolysable GTP analogue. If TD was applied to BLS in a divalent cation-containing buffer, the beta gamma-complex did not bind to the resin, whereas the alpha-subunit was retained; elution of the latter was achieved by removing the divalent cation from the buffer. Binding of the alpha-subunit to BLS was not affected by nucleotides or by ADP ribosylation catalysed by bacterial toxins. However, adsorption of the alpha-subunit by BLS or by a strong cation exchanger (Mono S) depended strictly on divalent cations. In contrast to previous reports, the data suggest the formation of a complex between a sulphonyl residue of Cibacron Blue, a divalent metal ion, and the alpha-subunit as the relevant binding mechanism causing adsorption of the alpha-subunit to BLS.

Animals↗
PubMed1987

Reversible activation of NADPH oxidase in membranes of HL-60 human leukemic cells.

NADPH oxidase in membranes of undifferentiated and dimethylsulphoxide-differentiated HL-60 cells was activated by arachidonic acid (AA) in the presence of Mg2+ and a cytosolic cofactor (CF) found in differentiated HL-60 cells. Basal superoxide (O2-) formation was enhanced several-fold by addition of the stable GTP-analogue, guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S), prior to AA and was completely prevented by that of GDP. Basal and GTP gamma S-stimulated O2- formation was terminated by GDP. In the presence of Mg2+ or EDTA, basal O2- formation ceased after 25 or 10 min, respectively, and was reinitiated by GTP gamma S or GTP gamma S plus Mg2+. Albumin terminated O2- formation, which was reactivated by AA in the presence of GTP gamma S. Our results show that (1) activation of NADPH oxidase in HL-60 membranes is dependent on endogenous GTP, Mg2+, AA and CF, which is induced during myeloid differentiation, and that (2) NADPH oxidase activation is a reversible process modulated by exogenous guanine nucleotides at various stages of activity of NADPH oxidase. We suggest crucial roles of guanine nucleotide-binding proteins in the activation, deactivation and reactivation of the enzyme.

Cell Differentiation↗
PubMed1987

In vitro synthesis of 32P-labelled phosphatidylinositol 4,5-bisphosphate and its hydrolysis by smooth muscle membrane-bound phospholipase C.

For studies of phospholipase C (PLC) activity in cell-free systems, 32P-labelled phosphatidylinositol 4,5-bisphosphate (PIP2) was prepared enzymatically by phosphorylating phosphatidylinositol 4-phosphate (PIP) in the presence of [gamma-32P]ATP using a PIP kinase partially purified from bovine retinae. PLC activity was determined by incubating membranes of DDT1 MF-2 cells with 32P-PIP2 and measuring remaining non-hydrolyzed substrate as well as accumulation of the hydrolysis product, inositol trisphosphate (IP3). Guanine nucleotides stimulated PIP2 hydrolysis and IP3 release. Additional increase in IP3 accumulation was observed with adrenaline plus guanine nucleotides.

Animals↗
PubMed1987

Fatty-acid-induced activation of NADPH oxidase in plasma membranes of human neutrophils depends on neutrophil cytosol and is potentiated by stable guanine nucleotides.

Both cis and trans unsaturated fatty acids and sodium dodecyl sulfate activated NADPH oxidase in plasma membranes of human neutrophils in the presence of neutrophil cytosol. In contrast, 5,8,11,14-icosatetraynoic acid, saturated fatty acids, esters, peroxides and 4 beta-phorbol 12-myristate 13-acetate, a potent activator of protein kinase C, were inactive. 5,8,11,14-icosatetraynoic acid inhibited superoxide formation elicited by fatty acids. Guanosine 5'[gamma-thio]triphosphate (GTP[gamma S]), a potent activator of guanine-nucleotide-binding proteins (N-proteins) enhanced superoxide formation elicited by fatty acids up to fourfold, supporting our previous suggestion that NADPH oxidase is regulated by an N-protein [Seifert, R. et al. (1986) FEBS Lett. 205, 161-165]. Cytosols from various tissues, soybean lipoxygenase and protein kinase C, purified from chicken stomach, did not substitute neutrophil cytosol. The activity of neutrophil cytosol was destroyed by heating at 95 degrees C. Superoxide formation was not affected by the inhibitor of protein kinase C 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7). Removal of cytosolic ATP by preincubation with hexokinase and glucose, dialysis of neutrophil cytosol or chelation of calcium with EGTA did not abolish the stimulatory effect of arachidonic acid and GTP[gamma S]. Thus, the cytosolic cofactor appears to be a neutrophil-specific and heat-labile protein, which is neither a lipoxygenase nor protein kinase C.

Cell Membrane↗
PubMed1987

NADP efficiently inhibits endogenous but not pertussis toxin-catalyzed covalent modification of membrane proteins incubated with NAD.

Incubation of membranes of human erythrocytes and platelets but not of human neutrophils with [32P]NAD leads to covalent modification of various membrane proteins and of added albumin. In membranes of all three cell types, pertussis toxin (PT), in the presence of NAD, specifically labelled a 40 kDa peptide, i.e. the alpha-subunit of a guanine nucleotide-binding protein. This effect of PT was slightly reduced by NADP, whereas modification of other membrane proteins and of albumin was largely suppressed, independent of whether PT was present or not. Labelling of cytosolic proteins in the presence of NAD was marginal; only in neutrophil cytosol, PT modified a 40 kDa peptide. Membranes of erythrocytes and platelets exhibited NAD-degrading activity, which was inhibited by NADP. The data suggest a high substrate specificity of PT for NAD. Inhibition of endogenous enzymes by NADP may prove useful for the evaluation of PT substrates.

Blood Platelets↗
PubMed1987

beta-Carotene Synthesis in Isolated Spinach Chloroplasts : Its Tight Linkage to Photosynthetic Carbon Metabolism.

Carefully isolated intact spinach chloroplasts virtually free of contamination of other organelles effectively form beta-carotene from NaH(14)CO(3) or [U-(14)C]-3-phosphoglycerate (PGA) under photosynthetic conditions. The photosynthate pool formed in chloroplasts from 1 to 2 millimolar [U-(14)C]-3-PGA or 3 to 6 millimolar NaH(14)CO(3) was fully sufficient to supply beta-carotene synthesis with intermediates for about 1 hour at maximal rates of about 20 nanomoles (14)C incorporated per milligram chlorophyll per hour. Fatty acid synthesis remains, under these circumstances, in linear dependence to substrate concentrations with far lower activity. Isotopic dilution of the beta-carotene synthesis by adding unlabeled glyceraldehyde 3-phosphate, dihydroxyacetone-P, 3-PGA, 2-PGA, phosphoenolpyruvate, pyruvate, respectively, may be interpreted as a direct substrate flow from photosynthetically fixed CO(2) to isopentenyl pyrophosphate synthesizing system. Unlabeled acetate did not dilute beta-carotene synthesis. Fatty acid synthesis acted similarly with unlabeled substrates; but it also was diluted by unlabeled acetate. These results indicate a tight linkage of photosynthetic carbon fixation and plastid isoprenoid synthesis.

Journal Article↗
PubMed1986

Guanine nucleotides stimulate NADPH oxidase in membranes of human neutrophils.

In the chain of events by which chemotactic peptides stimulate NADPH oxidase-catalyzed superoxide formation in human neutrophils, the involvements of a pertussis toxin-sensitive guanine nucleotide-binding protein (N-protein), mobilization of intracellular calcium and protein kinase C stimulation have been proposed. Superoxide formation was studied in membranes from human neutrophils; NADPH oxidase was stimulated by arachidonic acid in the presence of neutrophil cytosol. Fluoride and stable GTP analogues, such as GTP gamma S and GppNHp, which all activate N-proteins, enhanced NADPH oxidase activity up to 4-fold. GDP beta S inhibited the effect of GTP gamma S. These data suggest that NADPH oxidase is regulated by an N-protein, independent of an elevation of the cytoplasmic calcium concentration.

Cell Membrane↗
PubMed1986

Identification and characterization of the 35-kDa beta subunit of guanine-nucleotide-binding proteins by an antiserum raised against transducin.

Antisera were raised against the retinal guanine-nucleotide-binding protein (N-protein), transducin, purified from bovine rod outer segments. Sera obtained after repeated injections of antigen recognized all transducin subunits (alpha, beta and gamma). One antiserum, tested for cross-reactivity with non-retinal N-proteins, was found to cross-react with the beta subunits of the ubiquitously occurring N-proteins, Ns and Ni, but not with their respective alpha and gamma subunits. The antiserum also cross-reacted with the beta subunit of the recently identified N-protein, No, which has been found in high abundance in the central nervous system. These data support the similarity of the beta subunits of the N-proteins identified so far. Purification of N-proteins from porcine cerebral cortex without the use of activating ligands yielded fractions containing the isolated alpha subunit of No, free beta gamma complex, Ni, No and fractions containing both N-proteins in various proportions. The purity of the preparations was at least 80% as judged by Coomassie-blue-stained SDS gels. No pure Ns was obtained. Use of the transducin antibody during the course of the purification revealed that the beta subunits coeluted from a gel filtration column largely with the alpha subunits of Ni and No but were hardly detectable in fractions that were able to reconstitute Ns activity into membranes of an Ns-deficient cell line (S49 cyc- lymphoma cells). This indicates that in the central nervous system the concentrations of Ni and No are of magnitudes higher than that of Ns. Two-dimensional gel electrophoresis of N-proteins, purified from porcine cerebral cortex, resulted in the resolution of two major peptides in the 35-kDa region, which differed in their pI values and were identified as beta subunits by the use of the antiserum. Identical results were achieved using crude cholate extracts from membranes of the same tissue instead of purified proteins. The occurrence of different beta subunits may be explained by posttranslational N-protein modification.

Animals↗
PubMed1986

Vascular smooth muscle: availability of calcium through alpha-adrenoceptor stimulation.

In strip preparations from the rabbit main pulmonary artery, it was not possible to differentiate convincingly between alpha 1- and alpha 2-adrenoceptors, when selective agonists such as methoxamine, St 587, B-HT 920, and clonidine, as well as selective antagonists such as prazosin and yohimbine were used for receptor characterization. Measurements of the membrane potential of the vascular smooth-muscle cells with intracellular glass micropipettes showed similar depolarizations in response to the alpha 1-selective methoxamine and the alpha 2-selective B-HT 920. These observations suggest the occurrence of a uniform alpha-adrenoceptor population in the rabbit main pulmonary artery. However, in contrast to alpha 1-agonists, contractile responses to alpha 2-agonists differed considerably in their dependence on external Ca, as revealed in Ca withdrawal experiments, as well as by the use of Ca antagonists. Furthermore, inactivation of the guanine nucleotide-binding inhibitory protein (Ni protein) by pertussis toxin impaired vasoconstriction, in response to alpha 2-agonists, but left that to alpha 1-agonists unaffected. It is suggested that alpha 1- and alpha 2-agonists induce two distinct conformational changes in an otherwise uniform alpha-adrenoceptor of the rabbit main pulmonary artery. By the mediation of Ni protein, alpha 2-agonists appear to allow influx of Ca into the vascular muscle cells through a type of Ca channel that is unlikely to be controlled by voltage.

Adrenergic alpha-Agonists↗
PubMed1986

Peritoneal fluid volume, estrogen, progesterone, prostaglandin, and epidermal growth factor concentrations in patients with and without endometriosis.

Elevated prostaglandin (PG) levels in peritoneal fluid have been implicated as playing a role in infertility associated with endometriosis. This study was designed to measure peritoneal fluid levels of PG and other hormones that may influence PG release. Specific hormones measured included PGF2 alpha, PGE2, TxB2, 6-keto-PGF1 alpha, estrogen, progesterone, and epidermal growth factor. Peritoneal fluid volume and levels of estrogen, progesterone, and epidermal growth factor were significantly (P less than .05) increased during the secretory, as opposed to the proliferative, phase in both groups of patients, but no significant differences in these parameters were found between patients with and without endometriosis during either the proliferative or secretory phases. Although PG levels did not vary during the menstrual cycle in either group of patients, all four prostanoids were present in significantly (P less than .05) higher concentrations in patients with endometriosis as compared with patients without endometriosis. Furthermore, increased PG levels in patients with endometriosis appear to be due primarily to an increase in PG levels during the secretory phase of the cycle.

6-Ketoprostaglandin F1 alpha↗
PubMed1985

Extraction of the adenylate cyclase-activating factor of bovine sperm and its identification as a trypsin-like protease.

We previously reported the activation of adenylate cyclases from rat brain (Johnson, R. A., Awad, J. A., Jakobs, K. H., and Schultz, G., (1983) FEBS Lett. 152, 11-16) and from human platelets (Jakobs, K. H., Johnson, R. A., and Schultz, G. (1983) Biochim. Biophys. Acta 756, 369-375) by a factor derived from bovine sperm. In this report we describe the conditions for the extraction of the factor from bovine sperm and characteristics of its effects on adenylate cyclase which are consistent with its being a protease. The activating capacity of sperm particles was extracted from previously washed and frozen sperm into a 30,000 X g supernatant fraction by various salts, but not by the nonionic detergent Lubrol-PX. The amount of extracted factor: (a) was greatest with NH4HCO3 greater than NaCl greater than Na acetate; (b) was optimal with 0.5 M salt; (c) was not appreciably affected by the pH of the extraction buffer between pH 5.0 and 8.5; and (d) exhibited the greatest specific activity at the lower pH. The extracted sperm factor could be concentrated without loss by ultrafiltration on Amicon PM-10 membranes. The effect on adenylate cyclase of concentrated and desalted sperm extracted was inhibited 50% by various salts at 10 to 30 mM. The effects of the sperm factor to activate platelet adenylate cyclase, to block its inhibition via the alpha-adrenoceptor, and to block inhibition of stimulated forms of the enzyme by stable guanine nucleotides were prevented by protease inhibitors. A 50% reduction in the sperm factor's activation of platelet adenylate cyclase was caused by 30 nM soybean trypsin inhibitor, 30 nM alpha 2-macroglobulin, 300 nM leupeptin, 1 microM antipain, 15 microM aprotinin, and 100 microM benzamidine. Up to 3 mM phenylmethanesulfonyl fluoride was without effect on activation of the platelet cyclase by the sperm factor. The effects of the sperm factor persisted after its removal by the washing of pretreated platelet membranes and after its inactivation by the subsequent addition of leupeptin. The data strongly support the conclusion that the bovine sperm factor is a trypsin-like protease. alpha-Chymotrypsin, trypsin, and sperm acrosin were comparably effective in stimulating the platelet adenylate cyclase 5- to 8-fold, with concentrations eliciting maximal stimulation being: 200 ng trypsin/ml; 2 micrograms alpha-chymotrypsin/ml; and 2 micrograms acrosin/ml.(ABSTRACT TRUNCATED AT 400 WORDS)

Acrosin↗
PubMed1985

Localization and synthesis of prenylquinones in isolated outer and inner envelope membranes from spinach chloroplasts.

The prenylquinone content and biosynthetic capabilities of membrane fractions enriched in outer and inner envelope membranes from spinach chloroplasts were analyzed. Both envelope membranes contain prenylquinones, and in almost similar amounts (on a protein basis). However, the outer envelope membrane contains more alpha-tocopherol than the inner one although this prenylquinone is the major one in both fractions. On the contrary, plastoquinone-9 is present in higher amounts in the inner envelope membrane than in the outer one. In addition, it has been demonstrated that all the enzymes involved in the last steps of alpha-tocopherol and plastoquinone-9 biosynthesis, i.e., homogentisate decarboxylase polyprenyltransferase, S-adenosyl-methionine:methyl-6-phytylquinol methyltransferase, S-adenosyl-methionine: alpha-tocopherol methyltransferase, homogentisate decarboxylase solanesyltransferase, S-adenosyl-methionine:methyl-6-solanesylquinol methyltransferase, and possibly 2,3-dimethylphytylquinol cyclase, are localized on the inner envelope membrane. These results demonstrate that the inner membrane of the chloroplast envelope plays a key role in chloroplast biogenesis, and especially for the synthesis of the two major plastid prenylquinones.

Cell Fractionation↗