Parathyroid cell polarity as revealed by cytochemical localization of ATPases,alkaline phosphatase and 5′-nucleotidase

Summary Activities of Ca²⁺-dependent ATPase, Mg²⁺-dependent ATPase, Na⁺-K⁺-dependent ATP-ase, alkaline phosphatase, and 5-nucleotidase were demonstrated after incubation of 40-m vibratome sections of bovine parathyroids and subsequent visualization by electron microscopy. Prior to sectioning, parathyroid tissue was fixed with 1% glutaraldehyde for localization of alkaline phosphatase, and with 2% formaldehyde and 1% glutaraldehyde for demonstration activities of ATPases and 5-nucleotidase. The activities of the five enzymes were found at the apicolateral domain of the plasma membrane in parathyroid cells, i.e. at the site parathyroid cells face neighbouring parenchymal cells. Ca²⁺-ATPase activity was also seen on mitochondria, Golgi complex and RER. The presence of these plasma membrane associated enzymes at the apicolateral domain only indicate polarity in parathyroid cells. It further suggests that many processes including transmembrane transport take place at the apicolateral domain, the site of parathyroid cells opposing blood capillaries.     

Microscopy and cytochemistry of the biogenesis of the parasitophorous vacuole

Some parasitic protozoa are able to penetrate into host cells where they multiply. The process of penetration involves steps such as attachment to the host cell surface, internalization of the protozoan through an endocytic process with the formation of a parasitophorous vacuole (PV), and the subsequent interaction of the protozoan with the membrane lining the PV. This review analyzes the biogenesis of the PV from a morphological and cytochemical perspective. Special emphasis is given to (a) the localization of plasma membrane-associated enzymes such as Na⁺-K⁺-ATPase, Ca²⁺-ATPase, 5-nucleotidase, and NAD(P)H-oxidase, (b) glycoconjugates, detected using labeled lectins, (c) anionic sites, detected using cationic particles, and (d) integral membrane proteins, using freeze-fracture replicas, and lipids during the formation of the PV containing Trypanosoma cruzi, Leishmania, Toxoplasma gondii, and Plasmodium.     

Oxidative Inactivation of Brain Ecto-5′-Nucleotidase by Thiols/Fe2+ System

5-Nucleotidase, responsible for the conversion of adenosine-5-monophosphate into adenosine, was purified from bovine brain membranes, and subjected to oxidative inactivation. The 5-nucleotidase activity decreased slightly after the exposure to either glutathione or Fe²⁺. The glutathione-mediated inactivation of 5-nucleotidase was potentiated remarkably by Fe²⁺, but not Cu²⁺, in a concentration-dependent manner. Similarly, glutathione exhibited a concentration-dependent enhancement of the Fe²⁺-mediated inactivation. In comparison, the glutathione/Fe²⁺ system was much more effective than the ascorbate/Fe²⁺ system in inactivating the enzyme. In support of an intermediary role of superoxide ions or H₂O₂ in the action of glutathione/Fe²⁺ system, superoxide dismutase and catalase expressed a substantial protection against the inactivation by the glutathione/Fe²⁺ system. Meanwhile, hydroxyl radical scavangers such as mannitol, benzoate or ethanol were incapable of preventing the inactivation, excluding the participation of extraneous hydroxyl radicals. Whereas adenosine 5-monophosphate as substrate exhibited a modest protection against the glutathione/Fe²⁺ action, a remarkable protection was expressed by divalent metal ions such as Zn²⁺ or Mn²⁺. Structure-activity study with a variety of thiols indicates that the inactivating action of thiols in combination with Fe²⁺ resides in the free sulfhydyl group and amino group of thiols. Overall, thiols, expressing more inhibitory effect on the activity of 5-nucleotidase, were found to be more effective in potentiating the Fe²⁺- mediated inactivation. Further, kinetic analyses indicate that Fe²⁺ and thiols inhibit the 5-nucleotidase in a competitive or uncompetitive manner, respectively. These results suggest that ecto-5-nucleotidase from brain membrane is one of proteins susceptible to thiols/ Fe²⁺-catalyzed oxidation, and the oxidative inactivation may be related to the selective association of Fe²⁺ and thiols to the enzyme molecule.     

Phosphorylation of the plasma-membrane H+-ATPase of oat roots by a calcium-stimulated protein kinase

When plasma-membrane vesicles isolated from oat (Avena sativa L.) root cells were incubated with [-³²P]ATP, the H⁺-ATPase was found to be phosphorylated at serine and threonine residues. Phosphotyrosine was not detected. Endogenous ATPase kinase activity was also observed in plasma-membrane vesicles isolated from potato (Solanum tuberosum L.) root cells as well as from yeast (Saccharomyces cerevisiae). Identity of the phosphorylated oat root M_r=100 000 polypeptide as the ATPase was confirmed using conventional glycerol density-gradient centrifugation to purify the native enzyme and by a new procedure for purifying the denatured polypeptide using reversephase high-performance liquid chromatography. Kinase-mediated phosphorylation of the oat root plasma-membrane H⁺-ATPase was stimulated by the addition of low concentrations of Ca²⁺ and by a decrease in pH, from 7.2 to 6.2. These results demonstrate that kinase-mediated phosphorylation of the H⁺-ATPase is a plausible mechanism for regulating activity. They further indicate that changes in the cytoplasmic [Ca²⁺] and pH are potentially important elements in modulating the kinase-mediated phosphorylation.Abbreviations EDTA ethylenediaminetetraacetic acid - EGTA ethylene glycol-bis-(-aminoethyl ether)-N,N,N,N-tetraacetic acid - M_r relative molecular mass - RP-HPLC reverse-phase high-performance liquid chromatography - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis     

Isolation and characterization of the plasma membrane from Yoshida hepatoma cells

Summary Plasma membranes isolated from Yoshida ascites hepatoma AH-130 by a modification of the method of T. K. Ray (Biochim. Biophys. Acta 196: 1, 1970), were subfractionated into three fractions having densities (d) 1.12, 1.14 and 1.16 by discontinuous sucrose density-gradient. Membrane subfractions were characterized by electron-microscopy, by assay of marker enzymes and by lipid composition. All subfractions appeared to be essentially free from whole mitochondria, lysosomes and nuclei. Subfraction d 1.16 had, the highest 5-nucleotidase, Mg⁺⁺-ATPase and (Na⁺+K⁺)-ATPase activities; cytochromec oxidase was undetectable in any fraction and glucose-6-phosphatase was measurable only in fraction d 1.14. Adenylate cyclase had the highest activity in fractions d 1.14 and 1.16. Cyclic AMP phosphodiesterase was nearly equally distributed in the fractions. Adenylate, cyclase, 5-nucleotidase and Mg⁺⁺-ATPase activities of tumor membrane were lower with respect to liver plasma membrane, while cyclic AMP phosphodiesterase and (Na⁺+K⁺)-ATPase were found to have similar activities in the two membrane preparations. With respect to liver membrane, hepatoma membrane contained a higher amount of glycolipids and a higher amount of phospholipids accounted for mainly, by sphingomyelin, phosphatidylserine and phosphatidic acid. The possible significance of the decrease of adenylate activity in the hepatoma membrane is briefly discussed.     

Purification of the microsomal Ca2+-ATPase from rat liver

Summary The Ca²⁺-ATPase from rat liver microsomes has been solubilized in Triton X-100 and purified to homogeneity by ficollsucrose treatment, column chromatography with agarose-hexane adenosine 5-triphosphate Type 2, and high pressure liquid chromatography (HPLC). The purified enzyme obtained by this sequential procedure exhibited a 183-fold increase in specific activity. After ficoll-sucrose treatment, the activity of the Ca²⁺-ATPase was stable for at least two weeks when stored at –70°C. In SDS-polyacrylamide gels, several fractions from HPLC chromatography showed a single band at a position corresponding to a molecular weight of about 107 kDa. This value is consistent with the molecular weight of the phosphoenzyme intermediate of endoplasmic reticulum (ER) Ca²⁺-ATPase. Further characterization of the ER Ca²⁺-ATPase was performed by western immunoblots. Antiserum raised against the 100-kDa sarcoplasmic reticulum (SR) Ca²⁺-ATPase cross-reacted with the purified Ca²⁺-ATPase from rat liver ER membranes.     

Transmembrane Ca2+ gradient is essential for high anion transport activity of human erythrocytes

The role of a transmembrane Ca²⁺ gradient in anion transport by Band 3 of human resealed erythrocyte ghosts has been studied. The results show that a transmembrane Ca²⁺ gradient is essential for the conformation of erythrocyte Band 3 with higher anion transport activity. The dissipation of the transmembrane Ca²⁺ gradient by the ionophore A23187 inhibits the anion transport activity. The extent of this inhibition approaches 90% as the Ca²⁺ concentration on both sides of the ghost membrane is increased to 1.0 mM and half-maximum inhibition is observed at 0.25 mM Ca²⁺. Addition of ATP (0.4 mM) to the resealing medium can partly reestablish the transmembrane Ca²⁺ gradient by activation of Ca²⁺-ATPase and alleviate the inhibition to some extent. N-ethylmaleimide, an inhibitor of erythrocyte Ca²⁺-ATPase, prevents such restoration. Electron micrographs reveal that numerous larger intramembranous particles can be observed on the P-faces of freeze-fractured resealed ghosts in the absence of a transmembrane Ca²⁺ gradient.Abbreviations DPA dipicolinic acid - EITC eosin 5-isothiocyanate - DIDS 4,4-diisothiocyanostilbene-2,2-disulfonate - TES N-Tris-(hydroxymethyl)methyl-2-aminoethane sulfonic acid - PMSF phenylmethyl-sulfonylfluoride - NEM N-ethylamaleimide - BSA bovine serum albumin - EGTA ethyleneglycol-bis (aminoethylether)-tetra-acetic acid - EITC-Band 3 Band 3 labeled with EITC - Ca_i Ca²⁺ inside resealed ghosts - Ca_o Ca²⁺ outside resealed ghosts     

The mechanism of voltage-sensitive dye responses on sarcoplasmic reticulum

Summary The mechanism of voltage-sensitive dye responses was analyzed on sarcoplasmic reticulum vesicles to assess the changes in membrane potential related to Ca²⁺ transport. The absorbance and fluorescence responses of 3,3-diethyl-2,2-thiadicarbocyanine, 3,3-dimethyl-2,2-indodicarbocyanine and oxonol VI during ATP-dependent Ca²⁺ transport are influenced by the effect of accumulated Ca²⁺ upon the surface potential of the vesicle membrane. These observations place definite limitations on the use of these probes as indicators of ion-diffusion potential in processes which involve large fluctuations in free Ca²⁺ concentrations. Nile Blue A appeared to produce the cleanest optical signal to negative transmembrane potential, with least direct interference from Ca²⁺, encouraging the use of Nile Blue A for measurement of the membrane potential of sarcoplasmic reticulumin vivo andin vitro. 1,3-dibutylbarbituric acid (5)-1-(p-sulfophenyl)-3 methyl, 5-pyrazolone pentamethinoxonol (WW 781) gave no optical response during ATP-induced Ca²⁺ transport and responded primarily to changes in surface potential on the same side of the membrane where the dye was applied. Binding of these probes to the membrane plays a major role in the optical response to potential, and changes in surface potential influence the optical response by regulating the amount of membrane-bound dye. The observations are consistent with the electrogenic nature of ATP-dependent Ca²⁺ transport and indicate the generation of about 10 mV inside-positive membrane potential during the initial phase of Ca²⁺ translocation. The potential generated during Ca²⁺ transport is rapidly dissipated by passive ion fluxes across the membrane.     

Electrogenic behavior of the human red cell Ca2+ pump revealed by disulfonic stilbenes

Summary A systematic study was made of the action of 4-acetamido-4-isothiocyanostilbene-2,2-disulfonic acid (SITS) and 4,4-diisothiocyanostilbene-2,2-disulfonic acid (DIDS) on active Ca²⁺ transport of human erythrocytes. Pumping activity was estimated in inside-out vesicles (IOV's) by means of Ca²⁺-selective electrodes or use of tracer⁴⁵Ca²⁺. The stilbenes exhibited an approximately equal inhibitory potency and their action could be overcome by carbonyl cyanidep-trifluoromethoxyphenylhydrazone (FCCP) at low but not at high stilbene concentrations. In the absence of DIDS. Ca²⁺ transport was not affected upon addition of valinomycin, but it was appreciably reduced when vesicles were preincubated with low DIDS concentrations. Such an effect was strictly dependent on the external K⁺ concentration and it was abolished when valinomycin was added together with FCCP. Similar results were obtained using IOV's prepared from intact cells which had been previously exposed to the stilbene. The findings clearly demonstrate the presence in human red cells of a partially electrogenic Ca²⁺ pump, exchanging one Ca²⁺ ion for one proton.     

Inhibitory ca2+-control of movement of beads coated withphysarum myosin along actin-cables inChara internodal cells

Summary The actin-activated ATPase activityPhysarum myosin was shown to be inhibited of M levels of Ca²⁺. To determine if Ca²⁺ regulates ATP-dependent movement ofPhysarum myosin on actin, latex beads coated withPhysarum myosin were introduced intoChara cells by intracellular perfusion. In perfusion solution containing EGTA, the beads moved along the parallel arrays ofChara actin filaments at a rate of 1.0–1.8 m/sec; however, in perfusion solution containing Ca²⁺, the rate reduced to 0.0–0.7 m/sec. The movement of beads coated with scallop myosin, whose actin-activated ATPase activity is activated by Ca²⁺, was observed only in the perfusion solution containing Ca²⁺, indicating that myosin is responsible for the inhibitory effect of Ca²⁺ onPhysarum myosin movement. The involvement of this myosin-linked regulation in the inhibitory effect of Ca²⁺ on the cytoplasmic streaming observed inChara internodal cell andPhysarum plasmodium was discussed.Abbreviations ATP adenosine 5-triphosphate - DTT dithiothreitol - EDTA ethylenediaminetetraacetic acid - EGTA ethyleneglycolbis(-aminoethylether) N,N,N,N-tetraacetic acid - PIPES piperazine-N,N-bis(2-ethanesulfonic acid)     

IP3-and cAMP-induced responses in isolated olfactory receptor neurons from the channel catfish

Summary Olfactory receptor neurons enzymatically dissociated from channel catfish olfactory epithelium were depolarized transiently following dialysis of IP₃ or cAMP (added to the patch pipette) into the cytoplasm. Voltage and current responses to IP₃ were blocked by ruthenium red, a blocker of an IP₃-gated Ca²⁺-release channel in sarcoplasmic reticulum. In contrast, the responses to cAMP were not blocked by extracellularly applied ruthenium red, nor by l-cis-diltiazem or amiloride and two of its derivatives. The current elicited by cytoplasmic IP₃ in neurons under voltage clamp displayed a voltage dependence different from that of the cAMP response which showed marked outward rectification. A sustained depolarization was caused by increased cytoplasmic IP₃ or cAMP when the buffering capacity for Ca²⁺ of the pipette solution was increased, when extracellular Ca²⁺ was removed or after addition of 20–200 nm charibdotoxin to the bathing solution, indicating that the repolarization was caused by an increase in [Ca _i ] that opened Ca²⁺-activated K⁺ channels. The results suggest that different conductances modulated by either IP₃ or cAMP are involved in mediating olfactory transduction in catfish olfactory receptor neurons and that Ca²⁺-activated K⁺ channels contribute to the termination of the IP₃ and cAMP responses.Abbreviations ATP adenosine 5-triphosphate - BAPTA (bis-(o-aminophenoxy)-ethane-N-N-N-N)-tetraacetic acid - cAMP adenosine cyclic 3,5-monophosphate - cGMP guanosine cyclic 3,5-monophosphate - CTX charybdotoxin - DCB 3,4-dichlorobenzamil - EDTA ethylenediaminetetraacetic acid - EGTA ethylenglycol-bis-(b-aminoethyl)-N-N-N-N-tetraacetic acid - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - IP₃ inositol-1,4,5-triphosphate - NMDG N-methyl-d-glucamine We would like to thank the Tanabe Seiyaku Co., Ltd., for their gift of l-cis-diltiazem. This work was supported by National Institutes of Health grants DC00566 and BRSG S07RR05825.     

Study of some enzyme activities in cultured chick embryo brain nerve cells treated by chick embryo brain extracts

Brain extracts from 8-day-old chick embryos have been shown to influence morphological development of dissociated brain cells from 7-day-old chick embryos in culture. Stimulatory, effects on size of the neuronal somas and on growth of long processes were observed by adding the cytosol of the brain extract or the dialysate of the cytosol. These morphological changes parallel modifications of various enzyme activities according to the age of the cultures. Adenyl cyclase, (Na⁺, K⁺)- and Mg²⁺-ATPase, 5-nucleotidase, choline acetyltransferase, and acetylcholinesterase activities were studied between 5 and 14 days of culture. Adenyl cyclase activity was strongly stimulated at 8 days by both extracts. (Na⁺, K⁺)-and Mg²⁺-ATPase activities were stimulated in 8-day-old cultures only by the dialysate. 5-Nucleotidase activity was stimulated in 8-day-old cultures by the dialysate and in 11-day-old cultures by both extracts. Choline acetyltransferase activity was stimulated by the cytosol in 8-day-old cultures and by the dialysate in 11-day-old cultures. The total acetylcholinesterase activity was higher in 8-, 11-, and 14-day-old cultures treated with the cytosol. When the cells were treated with the dialysate, the activity was only higher in 14-day-old cultures. We also found that following the addition of brain extracts, the specific activity of the enzymes we studied was enhanced and became close to the values found in vivo during embryogenesis. Thus in parallel to the morphological modifications observed in nerve cell cultures treated by embryo brain extracts, biochemical variations especially involved in synaptogenesis and membrane development could be measured.     

The role of calcium ions in phytochrome-mediated germination of spores of Onoclea sensibilis L.

Phytochrome is confirmed to be the photoreceptor pigment in the germination response of Onoclea sensibilis L. by demonstrating red-far-red (R-FR) photoreversibility. External Ca²⁺ is required for this response with a threshold at a submicromolar concentration. Ethylene glycol-bis(-amino-ethyl ether)-N,N,N,N-tetraacetic acid, La³⁺ and Co²⁺ reversibly inhibit germination. Lanthanum only inhibits germination when applied before or during irradiation, indicating that the external Ca²⁺ requirement is transient, although in the absence of Ca²⁺ the R-stimulated system remains maximally poised to accept the ion for over 4 h after irradiation. The ability to respond to Ca²⁺ 4.1 h after R-irradiation is not reversed by FR-irradiation, indicating that Ca²⁺ transport has been uncoupled from phytochrome. Barium and Sr²⁺, but not Mg²⁺ can substitute for Ca²⁺. Artificially increasing the concentration of intracellular free Ca²⁺ with the ionophore A 23187 stimulates germination in the dark. The Ca²⁺-calmodulin antagonists, trifluoperizine and chlorpromazine, reversibly inhibit germination. Calcium is required in phytochrome-mediated fern spore germination; it may be acting as a second messenger.Abbreviations EGTA ethylene glycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid - FR far-red light - R fed light     

How do Ca2+ ions pass through the sarcoplasmic reticulum membrane

We propose an overview of the mechanism of Ca²⁺ transport through the sarcoplasmic reticulum membrane via the Ca²⁺-ATPase. We describe cytoplasmic calcium binding, calcium occlusion in the membrane and lumenal calcium dissociation. A channel-like structure is discussed and related to structural data on the membranous domain of the Ca²⁺-ATPase.Abbreviations SR Sarcoplasmic Reticulum - AMPPNP adenylyl-imidodiphosphate - AMPPCP adenylyl (,-methylene)-diphosphonate - FITC fluorescein 5-isothiocyanate - NBD 4-nitrobenzo-2-oxa-1,3-diazole - DCCD dicyclohexylcarbodiimide     

2,4,6-trinitrobenzenesulfonic acid modification of the carboxyl-terminal region (C-domain) of calreticulin

The role of the primary amino groups of lysine sidechains in Ca²⁺ binding to calreticulin was evaluated by chemical modification of the amino group with 2,4,6-trinitrobenzenesulfonic acid (TNBS). TNBS binding to calreticulin could be described by two steps: (i) a fast reaction, with low affinity, and (ii) a slow reaction with a relatively high affinity. Inclusion of Ca²⁺ and/or Mg²⁺ decreased both the amount of TNBS bound to calreticulin and the apparent affinity constant of the slower reaction. In contrast, the properties of the faster reaction for TNBS binding were not sensitive to Ca²⁺ and/or Mg²⁺. Analysis of TNBS binding to the carboxyl-terminal (C-domain) and aminoterminal (N-domain) of calreticulin revealed that theC-domain andN-domain are responsible for the slow and fast component of the TNBS binding, respectively. In keeping with this, in the presence of Ca²⁺, TNBS binding to theC-domain was significantly reduced, whereas modification of theN-domain was unaffected. TNBS modification of calreticulin significantly decreased Ca²⁺ binding to the low affinity/high capacity Ca²⁺ binding site(s) which are localized to theC-domain but had no effect on the high affinity/low capacity Ca²⁺ binding localized to theN-domain.In theC-domain of calreticulin, which contains the low affinity/high capacity Ca²⁺ binding sites, acidic residues are interspersed at regular intervals with one or more positively charged lysine and arginine residues. Our results indicate that the aminogroups of the lysine sidechains in theC-domain of calreticulin have a role in the low affinity/high capacity Ca²⁺ binding that is characteristic of this region of the protein and which is proposed to contribute significantly to the capacity of the endoplasmic reticulum Ca²⁺ store. (Mol Cell Biochem130: 19–28, 1994)Abbreviations TNBS 2,4,6-Trinitrobenzenesulfonic Acid - GST Glutathione S-Transferase - SDS-PAGE Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis - EDTA Ethylenediaminetetraacetic Acid - EGTA Ethylene Glycol bis(-aminoethylether)-N,N,N,N-tetraacetic Acid - MOPS 4-Morpholinepropanesulfonic Acid     

Mechanistic origin of the kinetic cooperativity for the ATPase activity of sarcoplasmic reticulum

The (Ca²⁺-Mg²⁺)-ATPase from sarcoplasmic reticulum presents negative cooperativity for the hydrolysis of Mg²⁺-ATP at different concentration ranges of this substrate. A kinetic model is proposed according to which Mg²⁺-ATP may bind to three different enzymatic species present during the catalytic cycle, E (K ₁=1 µM), EP.Ca₂ (K ₉=500 µM) and *EP (K ₇=20 µM), accelerating the release of P_i. The fact that each of these species has a different affinity for Mg²⁺-ATP allows a significant enhancement of the rate of P_i release to the medium at the different ranges of Mg²⁺-ATP concentration where the enzyme shows a kinetic cooperativity. The kinetic analysis of this mechanism yields an equation which is a ratio of two cubic polynomials (3:3 rate equations) with respect to Mg²⁺-ATP and which may explain the negative cooperativity of the enzyme at different concentration ranges of Mg²⁺-ATP.Abbreviations: EGTA, ethylene glycol bis(-aminoethylether)-N,N,N,N-tetraacetic acid; I.U., international units; piruvate kinase (EC 2.7.1.40); lactate dehydrogenase (EC 1.1.1.27); ATP phosphohydrolase (EC 3.8.1.3).     

Photolysis of caged Ca2+ induces trichocyst discharge inParamecium caudatum

Summary Trichocyst discharge, ciliary reversal, and cell body contraction inParamecium spp. have all been claimed to be regulated by the intracellular Ca²⁺ concentration ([Ca²⁺]_i) at the cortical region of the cell. We injected caged Ca²⁺ intoP. caudatum cells and applied ultraviolet (UV) light to the cell for 125 ms. This did not induce trichocyst discharge but did induce both ciliary reversal and cell body contraction. A re-application of UV for 125 ms triggered trichocyst discharge. These results demonstrate that (1) trichocyst discharge and ciliary reversal and cell body contraction are controlled by [Ca²⁺]_i and (2) the threshold of [Ca²⁺]i for trichocyst discharge is higher than those for the other two functions.Abbreviations DTT dithiothreitol - EGTA ethylene glycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid - ICL infraciliary lattice - [Ca²⁺]_i intracellular Ca²⁺ concentration - NP-EG o-nitrophenyl EGTA - PMT photomultiplier tube - UV ultraviolet     

Ca2+-binding sites and phosphatase activities in sieve element reticulum and P-protein of chick-pea phloem. A cytochemical and X-ray microanalysis survey

Summary In stem ofCicer arietinum, the loss of ribosomes attached to the rough ER cisternae during sieve element ontogeny results in the formation of sieve element reticulum (SER). By enhancing contrast of the SER, the OsFeCN postfixation/staining of material prefixed in glutaraldehyde in presence of calcium enables a good visualization of this membrane system. The pattern of staining in the SER is slightly lower when Mg²⁺ is substituted for Ca²⁺. These results support the view that the OsFeCN staining requires divalent cations and that the SER can accumulate Ca²⁺. The detection of Ca²⁺ by means of the pyroantimonate method in conjunction with X-ray microanalysis and the cytochemical localization of Ca²⁺ -ATPase in the SER cisternae provides evidence for Ca²⁺ sequestration by the SER. On the other hand, Ca²⁺-binding sites and ATPase activity are localized in P-protein. The ability to bind Ca²⁺ seems to enable the SER to function as an effective Ca²⁺ sink which may participate—with the sieve tube plasma membrane and mitochondria—in the maintenance of low Ca²⁺ concentration in phloem sap. In addition, the close association between P-protein and SER membranes exhibiting Ca²⁺-binding capabilities suggests that a Ca²⁺-mediated functional relationship may exist between the two structures. It is postulated that the SER may play a role in putative Ca²⁺ control of P-protein organization.Abbreviations SER sieve element reticulum - ER endoplasmic reticulum - P protein, phloem protein - OsFeCN method, osmium tetroxide-ferricyanide method - EDTA ethylenediamine tetraacetic acid - EGTA ethyleneglycol-bis-(-aminoethyl ether) N,N-tetraacetic acid - ATP adenosine 5-triphosphoric acid - ATPase adenosine triphosphatase - PCMB p chloromercuribenzoate - IDP inosine diphosphate     

Involvement of Ca2+-stimulated adenosine 5′-triphosphatase in the Ca2+ releasing mechanism of rat liver nuclei

The regulatory role of Ca²⁺-stimulated adenosine 5-triphosphatase (Ca²⁺-ATPase) in Ca²⁺ transport system of rat liver nuclei was investigated. Ca²⁺ uptake and release were determined with a Ca²⁺ electrode. Ca²⁺-ATPase activity was calculated by subtracting Mg²⁺-ATPase activity from (Ca²⁺–Mg²⁺)-ATPase activity. The release of Ca²⁺ from the Ca²⁺-loaded nuclei was evoked progressively after Ca²⁺ uptake with 1.0 mM ATP addition, while it was only slightly in the case of 2.0 mM ATP addition, indicating that the consumption of ATP causes a leak of Ca²⁺ from the Ca²⁺-loaded nuclei. The presence of N-ethylmaleimide (NEM; 0.1 mM) caused an inhibition of nuclear Ca²⁺ uptake and induced a promotion of Ca²⁺ release from the Ca²⁺-loaded nuclei. NEM (0.1 and 0.2 mM) markedly inhibited nuclear Ca²⁺-ATPase activity. This inhibition was completely blocked by the presence of dithiothreitol (DTT; 0.1 and 0.5 mM). Also, DTT inhibited the effect of NEM (0.1 mM) on nuclear Ca²⁺ uptake and release. Meanwhile, verapamil and diltiazem (10 M), a blocker of Ca²⁺ channels, did not prevent the NAD⁺ (1.0 and 2.0 mM), zinc sulfate (1.0 and 2.5 M) and arachidonic acid (10 M)-induced increase in nuclear Ca²⁺ release, suggesting that Ca²⁺ channels do not involve on Ca²⁺ release from the nuclei. These results indicates that an inhibition of nuclear Ca²⁺-ATPase activity causes the decrease in nuclear Ca²⁺ uptake and the release of Ca²⁺ from the Ca²⁺-loaded nuclei. The present finding suggests that Ca²⁺-ATPase plays a critical role in the regulatory mechanism of Ca²⁺ uptake and release in rat liver nuclei.     

Modulation of Phosphoenolpyruvate Carboxylase Phosphorylation in Leaves of Amaranthus hypochondriacus,a NAD-ME Type of C4 Plant

PEP carboxylase (PEPC) in leaves of C₄ plants is activated by phosphorylation of enzyme by a PEPC-protein kinase (PEPC-PK). We reevaluated the pattern of PEPC phosphorylation in leaf extracts of Amaranthus hypochondriacus. It was dependent on Ca²⁺, the optimum concentration of which for stimulation was 10 mM. The extent of stimulation was inhibited by 1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid (BAPTA), a Ca²⁺ chelator. The inhibition by BAPTA was relieved by the addition of Ca²⁺ but not by the addition of Mg²⁺. The stimulation by Ca²⁺ of PEPC phosphorylation was marginally enhanced by calmodulin (CaM), but not by diacylglycerol (DAG). Phosphorylation was strongly restricted by Ca²⁺ or Ca²⁺-CaM-dependent protein kinase inhibitors. Thus phosphorylation of PEPC is Ca²⁺-dependent in leaves of A. hypochondriacus and a calcium-dependent protein kinase (CDPK) may modulate PEPC-PK and subsequently the phosphorylation status of PEPC.