Cytokinins regulate a bidirectional phosphorelay network in Arabidopsis

The cytokinin class of plant hormones plays key roles in regulating diverse developmental and physiological processes. Arabidopsis perceives cytokinins with three related and partially redundant receptor histidine kinases (HKs): CRE1 (the same protein as WOL and AHK4), AHK2, and AHK3 (CRE-family receptors). It is suggested that binding of cytokinins induces autophosphorylation of these HKs and subsequent transfer of the phosphoryl group to a histidine phosphotransfer protein (HPt) and then to a response regulator (RR), ultimately regulating downstream signaling events. Here we demonstrate that, in vitro and in a yeast system, CRE1 is not only a kinase that phosphorylates HPts in the presence of cytokinin but is also a phosphatase that dephosphorylates HPts in the absence of cytokinin. To explore the roles of these activities in planta, we replaced CRE1 with mutant versions of the gene or with AHK2. Replacing CRE1 with CRE1(T278I), which lacks cytokinin binding activity and is locked in the phosphatase form, decreased cytokinin sensitivity. Conversely, replacing CRE1 with AHK2, which favors kinase activity, increased cytokinin sensitivity. These results indicate that in the presence of cytokinins, cytokinin receptors feed phosphate to phosphorelay-integrating HPt proteins. In the absence of cytokinins, CRE1 removes phosphate from HPt proteins, decreasing the system phosphoload.     

Cytokinins from a variant strain of cultured soybean cells

A strain of soybean cells capable of growing on a tissue culture medium lacking a cytokinin produced at least 3 compounds active in the soybean cytokinin assay. The characteristics of these compounds were consistent with their being zeatin in the free form, zeatin ribonucleoside and zeatin ribonucleotide. Although the conversion from a cytokinin dependent to independent condition in this strain parallels the change of normal cells to crown gall tumor state in terms of the capacity to synthesize cell division substances, the soybean factors are distinct from the nicotinamide derivatives reported for tumor cells of Vinca.     

Cytokinins and magnesium ions may control the flow of metabolites and calcium ions through fungal cell membranes

Compounds that are known to display cytokinin activity in green plants function as allosteric regulators of metabolite and ion transport in fungal cells. These hormonal compounds stimulate a massive release of calcium bound to a glycoprotein localised on the membrane surface, and simultaneously, activate the transport of calcium into the cells. The energy-linked import of sugars, nucleosides, and amino acids is inhibited by the cytokinins. The activating effect of cytokinins is neutralised by magnesium ions. Calcium and metabolite import in the fungi studied appear to depend upon a delicate balance between the concentrations of cytokinins, calcium, and magnesium ions.     

A new calcium binding glycoprotein family constitutes a major diatom cell wall component.

Diatoms possess silica-based cell walls with species-specific structures and ornamentations. Silica deposition in diatoms offers a model to study the processes involved in biomineralization. A new wall is produced in a specialized vesicle (silica deposition vesicle, SDV) and secreted. Thus proteins involved in wall biogenesis may remain associated with the mature cell wall. Here it is demonstrated that EDTA treatment removes most of the proteins present in mature cell walls of the marine diatom Cylindrotheca fusiformis. A main fraction consists of four related glycoproteins with a molecular mass of approximately 75 kDa. These glycoproteins were purified to homogeneity. They consist of repeats of Ca2+ binding domains separated by polypeptide stretches containing hydroxyproline. The proteins in the EDTA extract aggregate and precipitate in the presence of Ca2+. Immunological studies detected related proteins in the cell wall of the freshwater diatom Navicula pelliculosa, indicating that these proteins represent a new family of proteins that are involved in the biogenesis of diatom cell walls.     

Effect of trypsin on the cell surface proteins of hepatoma tissue culture cells. Characterization of a carbohydrate-rich glycopeptide released from a calcium binding membrane glycoprotein.

Concentrations of trypsin that bring about aggregation of hepatoma tissue culture (HTC) cells also release from the cell surface an Mr = 55,000 glycopeptide fragment. This glycopeptide fragment also accumulates in the medium, including serum-free medium, as a normal consequence of membrane protein turnover. The trypsin-released glycopeptide is labeled when cells are grown in the presence of fucose or leucine before treatment of the cells with the protease. Similarly, the glycopeptide fragment can be labeled by reacting cells in situ by lactoperoxidase-catalyzed radioiodination or by tritiated borohydride reduction of cells treated first with neuraminidase and galactose oxidase. The tryptic glycopeptide fragment was purified by concanavalin A-Sepharose chromatography, and hydroxyapatite chromatography in the presence of dodecyl sulfate. The amino acid and carbohydrate composition was determined, as was the sensitivity of the purified glycopeptide to a variety of endo- and exoglycosidases. The purified glycopeptide contains an average of 17 sialic acid residues and hence, shows charge heterogeneity after electrophoresis in isoelectric focusing gels. The charge heterogeneity can be eliminated completely by treatment with neuraminidase. The glycopeptide after this treatment is homogeneous. The trypsin-sensitive membrane glycoprotein which is the source of the Mr = 55,000 glycopeptide was identified by two-dimensional gel electrophoretic analysis of labeled cells, treated or not treated with trypsin. This glycoprotein, which has an apparent molecular weight of 85,000 and forms a homodimer in the presence of calcium ions, was purified and its identity as the parent of the Mr = 55,000 glycopeptide was confirmed by showing that the same Mr = 55,000 fragment was released by trypsin from the purified glycoprotein as was released from the intact cells.     

Critical sulfhydryls regulate calcium release from sarcoplasmic reticulum

Rapid Ca²⁺ release from the sarcoplasmic reticulum (SR) can be triggered by either binding of heavy metals to a sulfhydryl (SH) group or by catalyzing the oxidation of endogenous groups to a disulfide. Ca²⁺ release has been monitored directly using isolated vesicle preparations or indirectly by monitoring phasic contractions in a skinned fiber preparation. SH oxidation triggered by addition of Cu²⁺ /mercaptans, phthalocyanine dyes, reactive disulfides, and various anthraquinones appears to involve a direct interaction with the Ca²⁺ release protein from the SR. A model is presented in which reversible oxidation and reduction of endogenous SH groups results in the opening and closing of the Ca²⁺ release channel from the SR.Abbreviations SR sarcoplasmic reticulum - SH sulfhydryl - T-tubule transverse tubule - 2,2-DTDP 2,2-dithiodipyridine - 4,4-DTDP 4,4-dithiodipyridine - DTT dithiothreitol     

The glycoprotein nature and antigenicity of a fungal D-glucosyltransferase

D-Glucosyltransferase (EC 2.4.1.24) from Aspergillus niger has been prepared in pure form by chromatography on DEAE-cellulose. The enzyme transfers D-glucosyl units from maltose and other alpha-linked D-glucosyl oligosaccharides to glucosyl co-substrates resulting in the synthesis of new types of oligosaccharides. The glucosyltransferase has been found to be a glycoprotein containing 20% of carbohydrate consisting of mannose, glucose, and galactose. The carbohydrate residues are attached as either single units or as short oligosaccharide chains by O-glycosyl linkages to the serine and threonine residues of the protein. Antibodies directed against glucosyltransferase have been induced in animals by appropriate immunization regimes. These antibodies combine with the carbohydrate components of the enzyme and, therefore, the carbohydrate residues are the immunodeterminant groups of the glucosyltransferase.     

Hyaluronan and a cell-associated hyaluronan binding protein regulate the locomotion of ras-transformed cells

Hyaluronan (HA) and one of its cell binding sites, fibroblast hyaluronan binding protein (HABP), is shown to contribute to the regulation of 10T1/2 cell locomotion that contain an EJ-ras-metallothionein (MT-1) hybrid gene. Promotion of the ras-hybrid gene with zinc sulfate acutely stimulates, by 6-10-fold, cell locomotion. After 10 h, locomotion drops to two- to threefold above that of uninduced cells. Several observations indicate increased locomotion is partly regulated by HA. These include the ability of a peptide that specifically binds HA (HABR) to reduce locomotion, the ability of HA (0.001-0.1 micrograms/ml), added at 10-30 h after induction to stimulate locomotion back to the original, acute rate, and the ability of an mAb specific to a 56-kD fibroblast HABP to block locomotion. Further, both HA and HABP products are regulated by induction of the ras gene. The effect of exogenous HA is blocked by HABR, is dose-dependent and specific in that chondroitin sulfate or heparan have no significant effect. Stimulatory activity is retained by purified HA and lost upon digestion with Streptomyces hyaluronidase indicating that the activity of HA resides in its glycosaminoglycan chain. Uninduced cells are not affected by HA, HABR, or mAb and production of HA or HABP is not altered during the experimental period. These results suggest that ras-transformation activates an HA/HABP locomotory mechanism that forms part of an autocrine motility mechanism. Reliance of induced cells on HA/HABP for locomotion is transient and specific to the induced state.     

Cholecystokinin and insulin regulate insulin-like growth factor II binding to pancreatic receptors; Evidence of a role for intracellular calcium

The binding of ¹²⁵I-labeled insulin-like growth factor II (¹²⁵I-IGF II) to mouse pancreatic acini was stimulated (45%) by insulin and inhibited (30%) by cholecystokinin octapeptide (CCK₈). When CCK₈ and insulin were added together, the effect on IGF II binding was similar to that seen when CCK₈ was added alone. Two lines of evidence suggest that this effect of cholecystokinin on basal and insulin-stimulated ¹²⁵I-IGF II binding was mediated via a change in intracellular calcium: (1) the cholinergic agent carbachol inhibited IGF II binding to its receptors; (2) addition of the Ca²⁺ ionophore A23187 mimicked the effects of CCK₈ and carbachol. In contrast to its effects on IGF II binding to acini, CCK₈ had only small effects on IGF I binding and no effects on insulin binding.     

Isolation and characterization of Pseudomonas aeruginosa exotoxin A binding glycoprotein from mouse LM cells

A Pseudomonas aeruginosa exotoxin A (PE) binding glycoprotein was affinity purified from toxin sensitive mouse LM cells. The binding protein was solubilized with Triton X-100 or Nonidet P-40 and purified on a PE-Sepharose affinity column. Polyacrylamide gel electrophoresis yielded a single band with an estimated molecular mass of greater than 300,000 Da. N-Linked carbohydrate was present, accounting for approximately 10% of the total mass of the molecule. The purified protein specifically bound PE. Incubation of purified protein specifically bound PE. Incubation of purified PE binding protein with toxin reduced toxicity to LM cells. We speculate on the role of this toxin binding glycoprotein in the intoxication process.     

Analysis of calcium binding to alpha-lactalbumin using a fluorescent calcium indicator.

A sensitive and rapid assay of Ca2+ binding to proteins was developed, based on the competition of Ca2+ binding to the protein of interest and fluo-3, a fluorescent Ca2+ indicator. Ca2+ binding to fluo-3 and bovine alpha-lactalbumin was analyzed at ten different pH values and a range of Na+ and K+ concentrations. We demonstrate that the binding constants of alpha-lactalbumin, determined by means of the competition assay and using intrinsic protein fluorescence, are the same within experimental error. The dissociation constant of the alpha-lactalbumin--Ca2+ complex in 50 mM Hepes containing 150 mM Na+ at pH 7.4 and 25 degrees C, was found to be 123 +/- 2 nM and 103 +/- 43 nM when determined by the competition assay and intrinsic protein fluorescence, respectively. Binding of Ca2+ to alpha-lactalbumin did not depend on pH in the range 6.6-8.4 and was differently affected by Na+ and K+. EDTA-agarose, a chelating chromatography material, was synthesized and used to remove Ca2+ from buffer and protein solutions. The total concentration of Ca2+ in 50 mM Hepes, containing 150 mM Na+ at pH 7.4, was lowered to 119 +/- 13 nM and the number of Ca2+ bound/molecule alpha-lactalbumin was lowered to 0.069 +/- 0.006. No interaction between fluo-3 and alpha-lactalbumin could be discerned from spectral analysis and fluorescence anisotropy measurements.     

Intracellular calcium oscillations regulate ciliary beat frequency of airway epithelial cells

The effect of ATP-induced Ca2+ oscillations on ciliary activity was examined in airway epithelial cells by simultaneously measuring the ciliary beat frequency (CBF) and the intracellular Ca2+ concentration ([Ca2+]i) near the base of the cilia. Exposure to extracellular ATP (ATPo) induces a rapid and large increase in both [Ca2+]i and CBF, followed by oscillations in [Ca2+]i and a sustained elevation in CBF. After each Ca2+ oscillation, the [Ca2+]i returned to near basal values. By contrast, the CBF remained elevated during these Ca2+ oscillations, although each Ca2+ oscillation induced small variations in CBF. During Ca2+ oscillations, increases in CBF closely followed the rising phase of increases in [Ca2+]i, but declines in CBF lagged behind declines in [Ca2+]i. Higher frequency Ca2+ oscillations reduced variations in CBF, producing a stable and sustained elevation in CBF. The maximal CBF was induced by Ca2+ oscillations and was 15% greater than the CBF induced by the substantially larger initial [Ca2+]i increase. These data demonstrate that the rate of CBF is not directly dependent on the absolute [Ca2+]i, but is dependent on the differential changes in [Ca2+]i and suggest that CBF in airway epithelial cells is regulated by frequency-modulated Ca2+ signaling.     

Tamm-horsfall glycoprotein: a lectin binding study

Preparations of Tamm-Horsfall glycoprotein were examined by SDS-polyacrylamide gel electrophoresis and detection using various¹²⁵I-lectins as well as Coomassie Brilliant Blue. Considerable heterogeneity of electrophoretic pattern was seen. This was not due to a genetic polymorphism. Variation in binding by Soy-bean agglutinin was also seen. This was correlated with the Sd^a phenotype of the individual.     

Some properties of a glycoprotein with calcium binding ability found in human biological fluids in macroglobulinaemia IgM

An acidic glycoprotein with calcium-binding properties was isolated from the urine of patients with severe macroglobulinaemia IgM. The molecular weight of this protein determined by Sephadex gel filtration was found to be 62 000 ± 2800 in Tris · HCl buffer and 21 000 ± 1 000 in 6 M guanidine · HCl. The amino acid and carbohydrate composition of the isolated glycoprotein is presented. Electrophoretic migration of this protein was observed to be greatly affected by calcium ions present in the buffer in a concentration of 10^?3 M. At least two sets of binding sites seem to participate in binding calcium. The values 2.2 · 10⁶ M^?1 for the apparent association constant and 4.4 · 10^?4 mol of Ca²⁺ bound per g of protein for high affinity binding sites were estimated, on the basis of data from the equilibrium dialysis. The origin possible biological role of this protein is discussed.     

Intramolecular interactions regulate SAP97 binding to GKAP

Membrane-associated guanylate kinase homologs (MAGUKs) are multidomain proteins found to be central organizers of cellular junctions. In this study, we examined the molecular mechanisms that regulate the interaction of the MAGUK SAP97 with its GUK domain binding partner GKAP (GUK-associated protein). The GKAP-GUK interaction is regulated by a series of intramolecular interactions. Specifically, the association of the Src homology 3 (SH3) domain and sequences situated between the SH3 and GUK domains with the GUK domain was found to interfere with GKAP binding. In contrast, N-terminal sequences that precede the first PDZ domain in SAP97, facilitated GKAP binding via its association with the SH3 domain. Utilizing crystal structure data available for PDZ, SH3 and GUK domains, molecular models of SAP97 were generated. These models revealed that SAP97 can exist in a compact U-shaped conformation in which the N-terminal domain folds back and interacts with the SH3 and GUK domains. These models support the biochemical data and provide new insights into how intramolecular interactions may regulate the association of SAP97 with its binding partners.     

Exporting calcium from cells

All eukaryotic cells import Ca2+ through a number of variously gated plasma membrane channels. Once inside cells, Ca2+ transmits information to a large number of (enzyme) targets. Eventually, it must be exported again, to prevent the overloading of the cytosol with Ca2+. Two systems export Ca2+ from cells: a high affinity, low capacity Ca2+-ATPase, and a lower affinity, but much larger capacity, Na+/Ca2+ exchanger. The ATPase (commonly called the Ca2+ pump) is the fine-tuner of cell Ca2+, as it functions well even if the concentration of the ion drops below the microM level. It is a large enzyme, with 10 transmembrane domains and a C-terminal cytosolic tail that contains regulatory sites, including a calmodulin-binding domain. Four distinct gene products plus a large number of splice variants have been described. Some are tissue specific, the isoform 2 being specifically expressed in the sensorial cells of the Corti organ in the inner-ear. Its genetic absence causes deafness in mice. Two different families of the Na+/Ca2+ exchanger exist, one of which, originally described in photoreceptors, transports K+ and Ca2+ in exchange for Na+. The exchanger is particularly active in excitable cells, e.g., heart, where the necessity cyclically arises to rapidly eject large amounts of Ca2+. In addition to heart, the exchanger is particularly important to neurons: the cleavage of the most important neuronal isoform (NCX3) by calpains activated by excitotoxic treatments generates Ca2+ overload and eventually cell death.     

Visinin: a novel calcium binding protein expressed in retinal cone cells

Visinin is a retinal cone cell-specific protein (molecular weight 24,000, pI 5.1). To investigate its function, visinin cDNA was isolated from a chick retinal lambda gt11 cDNA library, using anti-visinin serum. The beta-galactosidase-visinin fusion protein was used for purifying epitope-selected antibody. The purified visinin antibody reacted only with a 24 kd protein in retinal cone cells. Visinin mRNA was expressed only in the retinal photoreceptor layer. The nucleotide sequence of the cDNA revealed that visinin has three E-F hand structures and is a Ca2+ binding protein. Visinin protein expressed in E. coli exhibited Ca2+ binding activity. These results suggest that visinin is a photoreceptor-specific Ca2+ binding protein and may be involved in phototransduction in the cone cells.