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D Marmé

Publications and source records attributed to D Marmé.

At least 91 records · Page 5Linked to original sources
PubMed1984

A Ca2+, Calmodulin-dependent NAD kinase from corn is located in the outer mitochondrial membrane.

NAD kinase activity from dark grown corn coleoptiles is shown to be almost totally dependent on Ca2+ and calmodulin. Nearly all of the enzyme activity is found in a particulate fraction. Upon differential and density gradient centrifugation the NAD kinase activity co-migrates with the mitochondrial cytochrome c oxidase whereas marker activities for nuclei, etioplasts, endoplasmic reticulum, and microbodies could well be separated, indicating that the NAD kinase is associated with mitochondria. This NAD kinase, associated with intact mitochondria, can be activated by exogenously added Ca2+ and calmodulin. In order to investigate the submitochondrial localization of the NAD kinase, the organelles were ruptured by osmotic treatment and sonication and the submitochondrial fractions were separated by density gradient centrifugation. The NAD kinase activity exhibits the same density pattern as the antimycin A-insensitive NADH-dependent cytochrome c reductase, a marker enzyme of the outer mitochondrial membrane. Marker enzymes for the mitochondrial matrix and the inner mitochondrial membrane reveal different density profiles. These results indicate that the Ca2+, calmodulin-dependent NAD kinase from coleoptiles of dark grown corn seedlings is located at the outer mitochondrial membrane. The physiological relevance of the location and the Ca2+, calmodulin-dependence of the NAD kinase will be discussed.

Calcium↗
PubMed1983

Calmodulin-stimulated cyclic nucleotide phosphodiesterase from Neurospora crassa.

Cyclic nucleotide phosphodiesterase has been partially purified by calmodulin-Sepharose affinity chromatography from a soluble extract of Neurospora crassa. The phosphodiesterase activity remained bound to the affinity column even in the presence of 6 M urea and could only be eluted by calcium chelation. The enzyme exhibits cAMP and cGMP phosphodiesterase activities. Both activities can be enhanced by calmodulin in a Ca2+-dependent manner. Stimulation of cyclic nucleotide phosphodiesterase by calmodulin can be inhibited by calmodulin antagonists such as pimozide, trifluoperazine and chlorpromazine.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1983

Myosin heavy chain kinase inactivated by Ca2+/calmodulin from aggregating cells of Dictyostelium discoideum.

Soluble myosin heavy chain kinases (MHC kinases) were partially purified from growth phase and aggregation-competent cells of Dictyostelium discoideum. In the aggregation-competent cells, two MHC kinases were distinguishable. One of these enzymes, called MHC kinase II, was inactivated by Ca2+ and calmodulin in a highly temperature-dependent reaction. A MHC kinase found in growth phase cells did not have these regulatory properties. Substrate specificities were analysed for MHC kinase II and for the MHC kinase from growth phase cells. Both enzymes phosphorylated threonine residues of the myosin heavy chains of D. discoideum and Physarum polycephalum. Phosphopeptide mapping of D. discoideum myosin and determination of the stoichiometry of its phosphorylation suggested the presence of two phosphorylation sites per heavy chain. Both sites were contained within a 38-kd chymotryptic fragment. The inactivation of MHC kinase II by Ca2+ plus calmodulin suggests this enzyme has a role in the regulation of myosin functions during the chemotactic response of a cell. The phosphorylated myosin had about one third the actin-activated Mg2+-ATPase activity of the non-phosphorylated myosin. Previous findings indicated that stimulation of D. discoideum cells with the chemo-attractant cAMP increases the cytoplasmic Ca2+ concentration. Under these conditions MHC kinase II might be inhibited and the dephosphorylated, more active form of myosin would accumulate.

Calcium↗
PubMed1983

Activation of plant quinate:NAD 3-oxidoreductase by Ca and calmodulin.

Quinate:NAD(+) 3-oxidoreductase (EC 1.1.1.24) from carrot cell suspension cultures has previously been shown to be activated by phosphorylation and inactivated by dephosphorylation. Here it is shown that the reactivation of the inactivated quinate:NAD(+) oxidoreductase is an enzyme-mediated process that requires ATP and protein kinase activity. The reactivation is completely inhibited by EGTA and can be restored by the addition of Ca(2+). Cyclic AMP at concentrations up to 5 muM did not have any effect on the reactivation either with or without EGTA in the medium. Calmodulin-depleted fractions containing quinate:NAD(+) oxidoreductase were obtained by passage of the crude extracts through an affinity column of 2-chloro-10-(3-aminopropyl)phenothiazine coupled to Sepharose 4B. The enzyme in this calmodulin-deficient fraction could be inactivated but not reactivated even in the presence of ATP and Ca(2+). However, addition of bovine brain calmodulin completely restored the activity of the enzyme. Half-maximal activation occurred at 130 nM calmodulin. We conclude from these data that the quinate:NAD(+) oxidoreductase is activated by a Ca(2+) - and calmodulin-dependent plant protein kinase.

Journal Article↗
PubMed1982

Calmodulin from neurospora crassa. General properties and conformational changes.

Calmodulin from Neurospora crassa has been purified to electrophoretic homogeneity. Equilibrium gel filtration experiments suggest that its Ca-binding properties are indistinguishable from those of vertebrate calmodulins. The isoelectric point of 4.04 and electrophoretic behavior under nondenaturing conditions indicate that N. crassa calmodulin is slightly less acidic than its vertebrate counterpart. The amino acid composition is typical of plant calmodulins with the exception that trimethyllysine is absent and that the content of Ser is unusually high. The tryptic peptide map of N. crassa calmodulin reveals an important number of point mutations as compared to vertebrate calmodulin. Differences in primary structure may explain why N. crassa calmodulin is less potent in the activation of myosin light chain kinase than calmodulins from higher organisms. The far UV circular dichroic spectra of the Ca-, Mg-, and metal-free forms of N. crassa calmodulin are similar to those of vertebrate calmodulin; in contrast, the near UV circular dichroic spectra are very different, apparently due to the differences in Tyr content. The single Tyr residue of N. crassa calmodulin, presumably located in position 138, undergoes an inversion of optical chirality upon addition of Ca2+, but not of Mg2+, to the metal-free protein.

Amino Acids↗
PubMed1980

Calmodulin activation of plant microsomal Ca uptake.

The active ATP-dependent uptake of Ca(2+) into a plasma membrane-enriched microsomal fraction from plants is stimulated by the addition of plant as well as bovine brain calmodulin. This stimulation occurs only when the microsomes are prepared in the presence of EDTA. Plant calmodulin also activates the bovine brain 3',5'-cyclic-nucleotide phosphodiesterase (EC 3.1.4.17). Mitochondrial Ca(2+) accumulation is not affected by calmodulin. The calmodulin-mediated activation of microsomal Ca(2+) uptake is abolished by the antipsychotic drug fluphenazine, whereas the calmodulin-independent Ca(2+) uptake is only slightly decreased.

Journal Article↗
PubMed1978

ATP-dependent Ca uptake into plant membrane vesicles.

Membrane vesicles were extracted from etiolated and light-grown plants, a plant cell suspension culture, and an alga. Upon addition of ATP and Mg(2+), active Ca(2+) uptake into the vesicles against a concentration gradient was shown. The dependence of this uptake on ATP and Mg(2+) concentrations, pH, and temperature is described. In the absence of oxalate, equilibrium between Ca(2+) uptake and efflux was reached after about 30 min. In the presence of oxalate, Ca(2+) accumulation continued for at least 120 min. Ca(2+) efflux from preloaded vesicles did not depend on ATP. Addition of the ionophores A23187 and Ro 20-0006/006 caused an immediate release of accumulated free Ca(2+). ATP-dependent Ca(2+) uptake was not inhibited by 10 muM oligomycin.

Journal Article↗
PubMed1976

Red Light-enhanced Phytochrome Pelletability: Re-examination and Further Characterization.

Red light-enhanced pelletability of phytochrome was observed in extracts of all 11 plants tested: Avena sativa L., Secale cereale L., Zea mays L., Cucurbita pepo L., Sinapis alba L., Pisum sativum L., Helianthus anuus L., Raphanus sativus L., Glycine max (L.) Merr., Phaseolus vulgaris L., and Lupinus albus L. This enhanced pelletability was observed in all 11 plants following in situ irradiation (in vivo binding) but only in Sinapis and Cucurbita after irradiation of crude extracts (in vitro binding). In vivo binding was not strongly dependent upon pH and, with few exceptions, was not markedly sensitive to high salt concentration, whereas in vitro binding was completely reversed by both high pH and high salt concentration. However, both binding phenomena were observed only with a divalent cation in the extract buffer. In vivo binding was further characterized using Avena which showed an increase in pelletability from less than 10% in dark control extracts to more than 60% in extracts of red light-irradiated shoots. The half-life for binding was 40 seconds at 0.5 C and was strongly temperature-dependent, binding being complete within 5 to 10 sec at 22 C. If pelletable phytochrome in the far red-absorbing form was photoconverted back to the red-absorbing form in situ, phytochrome was released from the pelletable condition with a half-life of 25 minutes at 25 C and 100 minutes at both 13 C and 3 C. No cooperativity in red light-enhanced pelletability with respect to phytochrome-far red-absorbing form was observed.

Journal Article↗
PubMed1974

The isolation and partial characterization of a membrane fraction containing phytochrome.

If 4-day-old dark-grown zucchini squash seedlings (Cucurbita pepo L. cv. Black Beauty) are exposed briefly to red light, subsequent cell fractionation yields about 40% of the total extractable phytochrome in the far red-absorbing form bound to a particulate fraction. The amount of far red-absorbing phytochrome in the pellet is strongly dependent on the Mg concentration in the extraction medium. The apparent density of the Pfr-containing particles following sedimentation on sucrose gradients corresponds to 15% (w/w) sucrose with 0.1 mm Mg and 40% sucrose with 10 mm Mg. This particulate fraction could be readily separated from mitochondria and other particulate material by taking advantage of these apparent density changes with changes in Mg concentration. Electron microscopy of negatively stained preparations shows that with 1 mm Mg only minute particles are present. These were too small to reveal structural detail with this technique. With 3 mm Mg, separate membranous vesicles between 400 and 600 Angstroms in diameter appear. At higher Mg concentrations, the vesicles aggregate, causing obvious turbity. The effect of Mg on vesicle formation and aggregation is completely reversible. Above 10 mm Mg, vesicle aggregation persists, but the percentage of bound Pfr decreases.

Journal Article↗
PubMed1974

In Vivo Properties of Membrane-bound Phytochrome.

After a 3-minute irradiation with red light, which saturates the phototransformation from the red light-absorbing form of phytochrome to the far red light absorbing form of phytochrome, about 40% of the phytochrome extractable from hooks of etiolated squash seedlings (Cucurbita pepo L. cv. Black Beauty) can be pelleted as Pfr at 17,000g after 30 minutes. Dark controls yield only 2 to 4% pelletable phytochrome in the form Pr. If a dark period intervenes between red irradiation and extraction, the bound Pfr gradually loses its photoreversibility. The time course for this destruction parallels the time course for phytochrome destruction in vivo following saturating red irradiation. The soluble fraction of phytochrome remains constant. These results suggest that in squash seedlings phytochrome destruction is related exclusively to the fraction which becomes membrane-bound. The induction of phytochrome binding by red light is not completely reversible by far red. In plants given saturating red followed immediately by saturating far red light, 12% of the phytochrome is found in the bound fraction as Pr if the phytochrome extraction is immediate. If a dark period intervenes between red-far red treatment and extraction, the bound phytochrome is released within 2 hours. A model of the binding properties of phytochrome, based on molecular interaction at the membrane is proposed, and possible consequences for the mechanism of action of phytochrome are discussed.

Journal Article↗
PubMed1973

Binding properties in vitro of phytochrome to a membrane fraction.

Brief irradiation of a buffer extract of dark-grown zucchini squash seedlings with red light results in the binding of the far-red-absorbing form of phytochrome to a particulate fraction. A low concentration of magnesium (0.1 mM) permits partial far-red reversal of the binding. A higher concentration (10 mM) yields actually enhanced binding after the far-red treatment. Both magnesium and calcium have a strong effect on the vesicularization of the phytochrome-binding particles and on their aggregation into readily sedimentable complexes. At concentrations above 10 mM, binding of the far-red-absorbing form of phytochrome is inhibited. These effects were not observed with sodium or potassium. Increasing the H(+) concentration led to increased binding of the far-red-absorbing form. This form of phytochrome bound at pH 6.5 and 10 mM magnesium is released if either the pH is raised to 8.0 or the magnesium concentration is raised to 50 mM. These properties suggest a new method for phytochrome purification.

Journal Article↗
PubMed1973

De novo synthesis of phytochrome in pumpkin hooks.

Phytochrome becomes density labeled in the hook of pumpkin (Cucurbita pepo L.) seedlings grown in the dark on D(2)O, indicating that the protein moiety of the pigment is synthesized de novo during development. Red light causes a rapid decline of the total phytochrome level in the hook of etiolated seedlings but upon return to the dark, phytochrome again accumulates. These newly appearing molecules are also synthesized de novo. Newly synthesized phytochrome in both dark-grown and red-irradiated seedlings is in the red-absorbing form. Turnover of the red-absorbing form is indicated by the density labeling of phytochrome during a period when the total phytochrome level in the hook of dark-grown seedlings remains constant. However, it was not possible to determine whether this results from intracellular turnover or turnover of the whole cell population during hook growth.

Journal Article↗