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V Niggli

Publications and source records attributed to V Niggli.

At least 55 records · Page 3Linked to original sources
PubMed1989

Diacylglycerols and PMA induce actin polymerization and distinct shape changes in lymphocytes: relation to fluid pinocytosis and locomotion.

Shape changes have been determined in human blood lymphocytes stimulated with OAG, diC8, PMA, colchicine or the hexapeptide fNLPNTL in short-term assays (30 min). Distinct types of shape-change responses were observed. Colchicine was active in generating a relatively small proportion of polarized lymphocytes (front-tail polarity). OAG, diC8 and PMA produced different types of shape change (non-polar cells with surface projections), and these were closely associated with an increase in actin polymerization and a shift of F-actin into the projections at the cell periphery. The diacylglycerols OAG and diC8 produced biphasic dose-response curves leading to rounding up of cells at very high stimulant concentrations. PMA produced no comparable biphasic response when tested over a much wider concentration range. Though the nonpolar cells with surface projections generated by OAG, diC8 or PMA showed vigorous shape changes, they lacked significant locomotor activity. alpha-Phorbol, 4 alpha-PDD, lumicolchicine or fNLPNTL were inactive. Small blood lymphocytes stimulated by OAG, diC8 or PMA showed a very small increase in the net uptake of FITC-dextran by fluid pinocytosis. Unlike neutrophils, which show a high net uptake, lymphocytes did not concentrate FITC-dextran in large granules, indicating that they do not develop a 'storage' compartment in the form of large vesicles. However, small fluorescent spots were consistently found in at least a fraction of blood lymphocytes. The results indicate that stimulated surface movement may be instrumental in fluid pinocytosis. Diacylglycerols may act as second messengers to induce pinocytosis, shape changes and altered actin polymerization in lymphocytes.

Actins↗
PubMed1989

Actin-associated proteins in human neutrophils: identification and reorganization upon cell activation.

The neutrophil cytoskeleton, especially the actin network, is thought to play a crucial role in neutrophil migration. However, little is known on the modulation of this network by actin-associated proteins. We have demonstrated the presence of immuno-reactive forms of alpha-actinin (an actin cross-linking and bundling protein) and vinculin (a putative actin-membrane linker) in human neutrophils using specific antibodies to chicken gizzard vinculin and bovine epithelial alpha-actinin. In contrast, talin, another putative actin-membrane linker protein, could not be detected in significant amounts in human neutrophils using a polyclonal antibody raised against chicken gizzard talin, which reacted with human platelet and lymphocyte talin. We have also analyzed the vinculin and alpha-actinin content of Triton X-100 insoluble cytoskeletons, isolated from resting and activated neutrophils. A small amount of alpha-actinin was already associated with the cytoskeleton of resting cells. Addition of chemotactic peptide to the cells rapidly increased the alpha-actinin content of the cytoskeletons 1.6 to 7-fold. This rapid increase was followed by a slower decrease to a lower level which, after 30 min of stimulation, was still significantly higher than that of control cells. The time-course of the association of alpha-actinin with the cytoskeleton paralleled that of actin association. This stimulus-induced rearrangement of cellular alpha-actin may thus play an important role in determining the structure of actin networks in motile neutrophils. Vinculin in contrast could not be detected in significant amounts in the Triton X-100-insoluble neutrophil cytoskeleton, not even after prolonged stimulation of the cells by chemotactic peptide.

Actinin↗
PubMed1986

Interaction of the cytoskeletal component vinculin with bilayer structures analyzed with a photoactivatable phospholipid.

The cytoskeletal component vinculin has been proposed to act as an actin-plasma membrane linker. In order to demonstrate a possible direct interaction of vinculin with bilayers, photolabeling with a phospholipid generating a highly reactive carbene was used. This phosphatidylcholine analogue (1-palmitoyl-2-[10-[4-[(trifluoromethyl)diazirinyl]phenyl]-[3H] 9-oxaundecanoyl]-sn-glycero-3-phosphocholine), with the photoactivatable diazirine group on its apolar portion, has been shown to label selectively membrane-embedded domains of membrane proteins. Vinculin is significantly labeled upon incubation and photolysis with liposomes containing trace amounts of this photoactivatable phospholipid, but only when the liposomes also contain acidic phospholipids. Labeling of vinculin is markedly increased (5-17-fold) by all acidic phospholipids tested so far (30%, w/w), compared to labeling in neutral phospholipids. Labeling is high at low ionic strength, but significant vinculin labeling can still be observed at physiological salt concentrations and acidic phospholipid content of the membrane. Our results provide evidence that vinculin inserts into the hydrophobic part of the bilayer by interacting with acidic phospholipids. A similar interaction may be of importance in vivo.

Actins↗
PubMed1984

Thrombin and activators of protein kinase C modulate secretory responses of permeabilised human platelets induced by Ca2+.

Addition of thrombin enhances secretion of both [14C]serotonin and beta-N-acetylglucosaminidase induced by Ca2+ in human platelets rendered permeable by exposure to intense electric fields. Enhancement of beta-N-acetylglucosaminidase secretion by thrombin results from an increase in the maximal extent of the response with no significant change in the median effective concentration EC50 for Ca2+. In contrast, thrombin shifts the dose/response curve for Ca2+-induced [14C]serotonin secretion to the left and has little effect on the maximal extent of this response even when this extent is reduced by use of a non-saturating concentration of MgATP2-. The relationship between extent of response and [MgATP2-] is similar for secretion of [14C]serotonin and of beta-N-acetylglucosaminidase in the presence or absence of thrombin. Similar nucleotide specificities are also observed. Activators of protein kinase C have previously been shown to mimic quantitatively the effect of thrombin on [14C]serotonin secretion induced by Ca2+ [D. E. Knight & M. C. Scrutton (1984) Nature (Lond.) 309, 66-68]. Such activators have the same qualitative effect as thrombin on the properties of beta-N-acetylglucosaminidase secretion induced by Ca2+ but are less effective. The EC50 for thrombin observed for enhancement of [14C]serotonin and beta-N-acetylglucosaminidase secretion is in the same range as that obtained for intact platelets under comparable conditions [D. E. Knight, T. J. Hallam & M. C. Scrutton (1982) Nature (Lond.) 296, 256-257]. The EC50, and the specificity of response, observed for activators of protein kinase C in these systems are consistent with those reported previously for the purified enzyme. Addition of 1-10 microM Ca2+ to permeabilised platelets in the presence of [gamma-32P] ATP causes marked enhancement of 32P incorporation into polypeptides of molecular mass 20 kDA, 45 kDA and 66 kDA. No additional polypeptides become phosphorylated in this system when thrombin is added together with 10 microM Ca2+, but some increase is observed in the extent of phosphorylation of the 45-kDa polypeptide. Addition of 1-oleyl-2-acetylglycerol + 1 - 2 microM Ca2+ causes enhanced phosphorylation of the 45-kDa polypeptide and to a lesser extent of the 20-kDa polypeptide. The dose/response curves for Ca2+-dependent phosphorylation of the 45-kDa polypeptide in the presence and absence of 1-oleyl-2-acetylglycerol are similar to those observed for Ca2+-dependent [14C]serotonin secretion under these conditions.(ABSTRACT TRUNCATED AT 400 WORDS)

Acetylglucosaminidase↗
PubMed1984

Tyrosine hydroxylase in "leaky" adrenal medullary cells: evidence for in situ phosphorylation by separate Ca2+ and cyclic AMP-dependent systems.

The systems responsible for phosphorylating tyrosine hydroxylase, the rate-limiting enzyme of catecholamine biosynthesis, were investigated in situ in adrenal medullary cells made permeable to solutes of up to 1,000 dalton by exposure to brief intense electric fields. Two different phosphorylation systems were found. One is dependent on Ca2+, the other on cyclic AMP. The Ca2+-dependent system is half-maximally activated by 1-2 microM Ca2+ and 0.5 mM ATP, and follows a time course similar to that of secretion of catecholamines. Trifluoperazine (0.1 mM) does not inhibit significantly Ca2+-dependent phosphorylation of tyrosine hydroxylase in situ. The cyclic AMP-dependent system is half-maximally activated by addition of 0.5 microM cyclic AMP and about 0.3 mM ATP. Ca2+-dependent and cyclic AMP-dependent phosphorylations of tyrosine hydroxylase have roughly the same time course and are additive under conditions where one system is already saturated. Peptide maps of immunoprecipitated tyrosine hydroxylase, after in situ phosphorylation of the enzyme either in the presence of 10(-8)M Ca2+ plus 2 X 10(-5)M cyclic AMP or of 10(-5)M Ca2+, show a marked difference indicating that the enzyme contains several phosphorylation sites. At least one of these sites is phosphorylated only by the Ca2+-dependent system, whereas the other site(s) are phosphorylated by both the Ca2+- and cyclic AMP-dependent systems. The effect of in situ phosphorylation of tyrosine hydroxylase on its enzymatic activity was also investigated.

Adrenal Medulla↗
PubMed1982

Molecular properties of calcium-pumping ATPase from human erythrocytes.

The Ca2+-pumping ATPase from human erythrocyte membranes, purified by the method previously reported [Niggli, V., Penniston, J. T., & Carafoli, E. (1979) J. Biol. Chem. 254, 9955-9958], was freed of minor impurities by extensive washing while bound to the calmodulin-Sepharose column. The pure enzyme showed a single band of Mr 138000, which contained no stainable carbohydrate. The enzyme retained calmodulin-stimulable ATPase activity; with appropriate assay conditions, an activity of 21.2 mumol/(mg x min) was obtained. Amino acid analysis showed that the ATPase had a larger proportion of polar amino acids than do other integral membrane proteins. Despite this, the ATPase showed a tendency to form dimers and higher aggregates even in the presence of sodium dodecyl sulfate and urea. The enzyme required Mg2+ but showed little activity unless a second ion was added. With regard to this second ion, the enzyme responded to alkaline earth metal ions in the order Ca2+ greater than Sr2+ much greater than Ba2+. It was highly specific for ATP and was stimulated by Na+ or K+; in all of these properties it resembled the enzyme in unfractionated membranes. Limited proteolysis using trypsin yielded, at short times, many fragments of various molecular weights; continued proteolysis resulted in two trypsin-resistant fragments of Mr 81000 and 33500. Analysis of the time course of proteolysis indicated that the ATPase existed in two or more conformations that had differing susceptibilities to proteolysis. It is suggested that these correspond to active and inactive conformers of the enzyme.

Amino Acids↗
PubMed1982

The Ca2+-pump of sickle cell plasma membranes. Purification and reconstitution of the ATPase enzyme.

The sickle cell (Hb SS) membrane-bound Ca2+-ATPase was found to have a Vmax in a range of 30-100% of the Vmax of the normal enzyme. In all sickle cell preparations, the Ca2+-ATPase could be stimulated at least 4-fold by calmodulin, but the stimulation factor varied considerably (4-26 fold) in the different preparations. The affinity of the ghost sickle cell Ca2+-ATPase for Ca2+, ATP and calmodulin was comparable to that of the normal enzyme. The sickle cell Ca2+-ATPase was solubilized from the membrane with Triton-X-100, and purified through a calmodulin sepharose-4B column, a technique by which the Ca2+-ATPase from normal ghosts has been successfully isolated in a functionally active and pure form (see V. Niggli, E.S. Adunyah, J.T. Penniston and E. Carafoli, 1981, J. Biol, Chem. 256, 395 - 401). The specific activity of the isolated sickle cell enzyme was significantly decreased (up to 80%) with respect to that ot the normal enzyme, but the amount of protein isolated was comparable to normal. All other parameters of the ATPase (affinity for Ca2+, ATP and calmodulin) were comparable to those found for the normal enzyme. In SDS polyacrylamide gel electrophoresis, the purified enzyme appeared as a single band protein with a Mr comparable to that of the normal enzyme. In the absence of calmodulin the sickle cell enzyme could be activated by acidic phospholipids, as reported for the normal enzyme. After reconstitution into liposomes it transported Ca2+ with normal efficiency (about 1 Ca2+/ATP hydrolyzed). Therefore, the only difference between the purified normal and the sickle cell enzyme appears to be the lower specific activity of the latter.

Adenosine Triphosphatases↗
PubMed1982

Purified red blood cell Ca2+-pump ATPase: evidence for direct inhibition by presumed anti-calmodulin drugs in the absence of calmodulin.

A variety of presumed anti-calmodulin (anti-CaM) drugs was tested for their potential inhibitory effects on the isolated, purified and reconstituted Ca2+-pump ATPase of human red blood cell membranes. Anti-CaM drugs inhibited the Ca2+-pump ATPase both in the absence and presence of added CaM. Qualitatively similar inhibition was observed in two different ATPase assay systems. In asolectin vesicles in the absence of added CaM trifluoperazine (TFP), N-(6-aminohexyl)-5-chloro-1-naphthalene- sulfonamide (W-7), vinblastine, dibucaine, imipramine, propranolol and dimethylpropranolol (UM-272) were all inhibitory. Potency of anti-CaM drugs was generally greater on the enzyme reconstituted in asolectin vesicles than on the enzyme reconstituted in phosphatidylcholine vesicles, either in the presence or absence of CaM. The results emphasize that anti-CaM drugs have actions other than to bind to CaM. Possible direct interaction of amphipathic cationic anti-CaM drugs with the Ca2+-pump ATPase and/or its lipid environment is suggested.

Calcium-Binding Proteins↗
PubMed1981

Acidic phospholipids, unsaturated fatty acids, and limited proteolysis mimic the effect of calmodulin on the purified erythrocyte Ca2+ - ATPase.

The purified Ca2+-pumping ATPase of human erythrocyte membranes (Niggli, V., Adunyah, E. S., Penniston, J. T., and Carafoli, E. (1981) J. Biol. Chem. 256, 395-401) can be stimulated, in the absence of calmodulin, by other treatments. 1. A variety of acidic phospholipids (phosphatidylserine, cardiolipin, phosphatidylinositol, and phosphatidic acid) stimulate the Vmax and decrease the Km (Ca2+) of the isolated enzyme to the same extent as calmodulin. Unsaturated fatty acids (oleic and linoleic acid) have the same effect as phospholipids but at lower concentrations. Neutral phospholipids (phosphatidylcholine, sphingomyelin, and phosphatidylethanolamine) have no effect on the enzyme. The minimal proportion of acidic phospholipids in the environment of the enzyme necessary for full stimulation is about 40%. 2. The isolated enzyme, after reconstitution in phosphatidylcholine liposomes in the absence of calmodulin, can be activated by limited proteolysis. The trypsinized enzyme has the same high Vmax and high affinity for Ca2+ of the enzyme in the presence of calmodulin.

Calcium-Binding Proteins↗
PubMed1981

Purified (Ca2+-Mg2+)-ATPase of the erythrocyte membrane. Reconstitution and effect of calmodulin and phospholipids.

The Ca2+-pumping ATPase from human erythrocyte membranes, purified nearly to homogeneity (Niggli, V., Penniston, J. T., and Carafoli, E. (1979) J. Biol. Chem. 254, 9955-9958), can be reconstituted into phospholipid vesicles. The purified and the reconstituted forms of the enzyme displayed the properties expected of the intact Ca2+ pump; they had an appropriate (Ca2+-Mg2+)-ATPase activity which displayed a relatively low affinity for Ca2+. Added calmodulin increased both the maximum rate and the affinity for Ca2+ of the enzyme. Mg2+ alone caused no significant ATP hydrolysis in the purified enzyme, indicating that the Mg2+-ATPase is a separate enzyme. Vesicles of the reconstituted enzyme accumulated Ca2+ with a ratio of Ca2+ accumulated to ATP hydrolyzed of approximately 1. Ca2+ accumulation and ATPase of the reconstituted enzyme were inhibited concurrently by vanadate ion, with a K 1/2 for inhibition which was indistinguishable from that observed for the (Ca2+-Mg2+)-ATPase in whole erythrocyte ghosts. While the above properties were all consistent with those observed for the (Ca2+-Mg2+)-ATPase in whole erythrocyte ghosts, the purified enzyme displayed an unexpected response to acidic phospholipids. Enzyme reconstituted with or prepared in phosphatidylserine acted as if calmodulin were already present, and added calmodulin caused no effect beyond that due to phosphatidylserine. This mimicry of the calmodulin effect by acidic phospholipids is similar to that reported for cyclic nucleotide phosphodiesterase (Wolff, D. J., and Brostrom, C. O. (1976) Arch. Biochem. Biophys., 173, 720-723).

Biological Transport, Active↗
PubMed1981

Interaction of the purified Ca2+, Mg2+-ATPase from human erythrocytes with phospholipids and calmodulin.

The Ca2+-pumping ATPase has been purified in a functional form from human erythrocytes by calmodulin affinity chromatography. The purified enzyme has a specific activity at least 300-fold higher than the membrane bound enzyme. It consists of one major protein band of 140000 Dalton, and after reconstitution in liposomes it transports Ca2+ with an efficiency of at least 1 Ca2+/ATP. In the presence of calmodulin, the affinity of the enzyme for Ca2+, and its specific activity, are greatly increased. Acidic phospholipids have an unexpected effect on the isolated enzyme: ATPase isolated or reconstituted in acidic phospholipids behaves as if calmodulin were present. Acidic phospholipids mimic the effect of calmodulin.

Ca(2+) Mg(2+)-ATPase↗