Divalent cation stimulation of in vitro fibre assembly from epidermal keratin protein

1978 ◽  
Vol 33 (1) ◽  
pp. 255-263
Author(s):  
K. Fukuyama ◽  
T. Murozuka ◽  
R. Caldwell ◽  
W.L. Epstein
Keyword(s):  
Ionic Strength ◽  
Divalent Cation ◽  
Divalent Cations ◽  
Urea Concentration ◽  
Newborn Rats ◽  
Filament Formation ◽  
Stimulation Of ◽  
Millimolar Concentration

Keratin was extracted from purified cornified cells of newborn rats in Tris-HCl-buffered 8 M urea containing beta-mercaptoethanol. Microfilaments were assembled in vitro by reducing the ionic strength of buffer and the urea concentration. One millimolar concentration of KCl and NaCl did not affect filament formation, but the same concentration of divalent cations greatly altered this process. CaCl2 and MgCl2 induced gelation of keratin by formation of bundles of birefringent macrofilaments. ZnCl2, CuSO4 and HgCl2 formed greater numbers of macrofilaments and the protein aggregated.

Filament Formation by Slime Mould Myosin Isolated at Low Ionic Strength

1974 ◽  
Vol 15 (1) ◽  
pp. 113-129
Author(s):  
H. HINSSEN ◽  
J. D'HAESE
Keyword(s):  
Ionic Strength ◽  
Striated Muscle ◽  
Divalent Cations ◽  
Filament Formation ◽  
Selective Precipitation ◽  
Slime Mould ◽  
Preparation Conditions ◽  
Filament Structure ◽  
Low Ionic Strength

Myosin was isolated and purified from plasmodia of the slime mould Physarum polycephalum by a new method. This method is based on actomyosin extraction at low ionic strength after extensive washing, followed by the selective precipitation of myosin at pH 6.1 under relaxing conditions. The yield of myosin was 3-5 times higher than reported for other methods. In contrast to earlier studies a remarkably strong tendency to filament formation was found for slime mould myosin, probably due to a better preservation of some structural properties during preparation. Conditions were worked out under which numerous filaments up to 4 µm in length can be produced. It was established that not only a gradual decrease of ionic strength may influence filament formation, but also pH, ATP concentration and the presence of divalent cations. Compared to the current filament models a difference exists in the structure of the filaments. No central bare zone can be found, and thus, they lack an apparent bipolarity. Along the entire filament there are lateral projections representing the head portion of myosin molecules. A clear periodicity with an axial repeat of about 14 nm was observed, indicating a highly ordered arrangement of these projections. In this paper it is shown for the first time that myosin from one of the primitive motile systems is able to form aggregates of high structural order, indicating that the contraction of non-muscular actomyosin systems is not necessarily effected with oligomeric or randomly aggregated myosin. The possible role of myosin aggregation in vivo and the similarity of filament structure to that recently reported for myosin from vertebrate smooth muscle and striated muscle are discussed.

scholarly journals Stimulation of ribonucleic acid polymerase activity in vitro by prostatic steroid–protein receptor complexes

1973 ◽  
Vol 136 (3) ◽  
pp. 611-622 ◽  
Author(s):  
P. Davies ◽  
K. Griffiths
Keyword(s):  
Ionic Strength ◽  
Rna Polymerase ◽  
Polymerase Activity ◽  
Supernatant Fraction ◽  
Nuclear Fraction ◽  
Receptor Complexes ◽  
Protein Receptor ◽  
Ammonium Sulphate Fractionation ◽  
Stimulation Of

A system has been developed which allows the stimulation in vitro of prostatic RNA polymerase by prostatic 5α-dihydrotestosterone–protein receptor complexes prepared from the tissues of castrated rats. The reconstitution in vitro of such a system necessitates the purification of several subcellular components. Two 5α-dihydrotestosterone–receptor complexes are located in the prostatic soluble supernatant fraction, separable by selective ammonium sulphate fractionation, and one complex can be isolated from the nuclear fraction. In the presence of all these complexes, stimulation of RNA polymerase in intact nuclei and nucleoli was observed. The complexes also increased the activity of the enzyme solubilized from whole nuclei. Greater stimulation of this system was noted in the presence of prostatic chromatin as template, as compared with that observed with calf thymus DNA or liver chromatin as template. The effects of the complexes on subnuclear forms of RNA polymerase, of nucleolar and extranucleolar origin, are also described. RNA polymerase solubilized from nucleoli is more susceptible to stimulation by the 5α-dihydrotestosterone–receptor complexes than is the ‘nucleoplasmic’ enzyme. Stimulation occurs less readily in the presence of Mn2+and at high ionic strength than in the presence of Mg2+and at low ionic strength. Preliminary experiments show that prostatic nucleolar RNA polymerase transcribes prostatic chromatin poorly as compared with the nucleoplasmic enzyme. The observations reported indicate an involvement of non-histone proteins associated with DNA in the process by which stimulation of enzyme activity by the 5α-dihydrotestosterone–receptor complexes is achieved. The implications of these findings in the mechanism of steroid hormone action is considered.

scholarly journals Characterization of the microtubule-activated ATPase of brain cytoplasmic dynein (MAP 1C).

1988 ◽  
Vol 107 (3) ◽  
pp. 1001-1009 ◽  
Author(s):  
H S Shpetner ◽  
B M Paschal ◽  
R B Vallee
Keyword(s):  
Ionic Strength ◽  
Divalent Cations ◽  
High Sensitivity ◽  
Retrograde Transport ◽  
Cytoplasmic Dynein ◽  
Maximum Activity ◽  
Scanning Transmission ◽  
Sulfhydryl Oxidation ◽  
The Brain

We recently found that the brain cytosolic microtubule-associated protein 1C (MAP 1C) is a microtubule-activated ATPase, capable of translocating microtubules in vitro in the direction corresponding to retrograde transport. (Paschal, B. M., H. S. Shpetner, and R. B. Vallee. 1987b. J. Cell Biol. 105:1273-1282; Paschal, B. M., and R. B. Vallee. 1987. Nature [Lond.]. 330:181-183.). Biochemical analysis of this protein (op. cit.) as well as scanning transmission electron microscopy revealed that MAP 1C is a brain cytoplasmic form of the ciliary and flagellar ATPase dynein (Vallee, R. B., J. S. Wall, B. M. Paschal, and H. S. Shpetner. 1988. Nature [Lond.]. 332:561-563). We have now characterized the ATPase activity of the brain enzyme in detail. We found that microtubule activation required polymeric tubulin and saturated with increasing tubulin concentration. The maximum activity at saturating tubulin (Vmax) varied from 186 to 239 nmol/min per mg. At low ionic strength, the Km for microtubules was 0.16 mg/ml tubulin, substantially lower than that previously reported for axonemal dynein. The microtubule-stimulated activity was extremely sensitive to changes in ionic strength and sulfhydryl oxidation state, both of which primarily affected the microtubule concentrations required for half-maximal activation. In a number of respects the brain dynein was enzymatically similar to both axonemal and egg dyneins. Thus, the ATPase required divalent cations, calcium stimulating activity less effectively than magnesium. The MgATPase was inhibited by metavandate (Ki = 5-10 microM for the microtubule-stimulated activity), 1 mM NEM, and 1 mM EHNA. In contrast to other dyneins, the brain enzyme hydrolyzed CTP, TTP, and GTP at higher rates than ATP. Thus, the enzymological properties of the brain cytoplasmic dynein are clearly related to those of other dyneins, though the brain enzyme is unique in its substrate specificity and in its high sensitivity to stimulation by microtubules.

scholarly journals Effects of α-amanitin on the stimulation of prostatic ribonucleic acid polymerase by prostatic steroid–protein receptor complexes

1974 ◽  
Vol 140 (3) ◽  
pp. 565-567 ◽  
Author(s):  
P. Davies ◽  
K. Griffiths
Keyword(s):  
Ionic Strength ◽  
Rna Polymerase ◽  
Ribonucleic Acid ◽  
Selective Inhibitor ◽  
High Ionic Strength ◽  
Receptor Complexes ◽  
Protein Receptor ◽  
Stimulation Of

Stimulation of prostatic RNA polymerase in vitro by prostatic 17β-hydroxy-5α-androstan-3-one (5α-dihydrotestosterone)–receptor complexes has been previously reported. By use of the selective inhibitor, α-amanitin, we have shown that both nucleolar and extranucleolar RNA polymerase activities may be stimulated, but stimulation is abolished at high ionic strength.

Regulatory effects of potassium and inorganic anions on the NADP-specific malic enzyme of Escherichia coli

1985 ◽  
Vol 63 (2) ◽  
pp. 128-136
Author(s):  
Deborah A. Brown ◽  
Robert A. Cook
Keyword(s):  
Escherichia Coli ◽  
Ionic Strength ◽  
Binding Sites ◽  
Malic Enzyme ◽  
Divalent Cations ◽  
Kinetic Studies ◽  
Regulatory Effects ◽  
Metal Cofactors

The effects of K+ and various anions on the catalytic and regulatory properties of the NADP-specific malic enzyme of Escherichia coli are reported. Studies on the susceptibility of the enzyme to proteolysis indicate that K+ binds directly to the enzyme with a resultant change in enzyme conformation. Kinetic studies indicate that the binding of optimal concentrations of K+ results in activation of the enzyme, increasing both the Vmax and the affinity of the enzyme for divalent cations. The inhibition of enzyme activity observed at KCl concentrations greater than 50 mM is shown to be nonspecific, resulting from increasing ionic strength. The mixed cooperativity between malate-binding sites previously reported at optimal K+ concentration is more pronounced at nonoptimal K+ concentrations (0 and 150 mM). The regulatory effect of metal cofactors and the mixed cooperativity between malate-binding sites is abolished when kinetic studies are conducted at low ionic strength or in the presence of acetate. Acetate appears to act as an activator, increasing the affinity of the enzyme for malate and protecting the enzyme against the inhibition caused by high ionic strength. It is postulated that the enzyme is operating in vivo in a partially inhibited state owing to the ionic strength of the cytoplasm. The kinetic studies conducted at higher ionic strength in vitro are therefore more applicable to the in vivo situation.

scholarly journals Organization in the cell nucleus: divalent cations modulate the distribution of condensed and diffuse chromatin.

1981 ◽  
Vol 90 (1) ◽  
pp. 181-186 ◽  
Author(s):  
R P Aaronson ◽  
E Woo
Keyword(s):  
Cell Nucleus ◽  
Divalent Cation ◽  
Divalent Cations ◽  
Chromatin Condensation ◽  
Ultrastructural Organization ◽  
Organizational Changes ◽  
Nuclear Ultrastructure ◽  
Liver Nuclei ◽  
Inactive Chromatin

The organization of rat liver nuclei in vitro depends on the ionic milieu. Turbidity measurements of nuclear suspensions in the presence of varying concentrations of divalent cations have been correlated with nuclear ultrastructure. The concentration of MgCl2 (2 mM) at which turbidity of nuclear suspensions is maximal and chromatin condensation appears most extensive is the same concentration that reportedly (Gottesfeld et al., 1974, Proc. Natl. Acad. Sci. U. S. A. 71:2193-2197) precipitates "inactive" chromatin. Thus, a mechanism is suggested by which chromatin activity and ultrastructural organization within the nucleus may be mediated. The nuclear organizational changes attendant upon the decrease in divalent cation concentration were not entirely reversible.

scholarly journals Synaptotagmin Isoforms Couple Distinct Ranges of Ca2+, Ba2+, and Sr2+ Concentration to SNARE-mediated Membrane Fusion

2005 ◽  
Vol 16 (10) ◽  
pp. 4755-4764 ◽  
Author(s):  
Akhil Bhalla ◽  
Ward C. Tucker ◽  
Edwin R. Chapman
Keyword(s):  
Membrane Fusion ◽  
Synaptic Vesicles ◽  
Binding Protein ◽  
Divalent Cations ◽  
Attachment Protein ◽  
Protein Receptor ◽  
Sensitive Factor ◽  
Factor Attachment Protein Receptor ◽  
Stimulation Of

Ca2+-triggered exocytosis of synaptic vesicles is controlled by the Ca2+-binding protein synaptotagmin (syt) I. Fifteen additional isoforms of syt have been identified. Here, we compared the abilities of three syt isoforms (I, VII, and IX) to regulate soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)-mediated membrane fusion in vitro in response to divalent cations. We found that different isoforms of syt couple distinct ranges of Ca2+, Ba2+, and Sr2+ to membrane fusion; syt VII was ∼400-fold more sensitive to Ca2+ than was syt I. Omission of phosphatidylserine (PS) from both populations of liposomes completely abrogated the ability of all three isoforms of syt to stimulate fusion. Mutations that selectively inhibit syt·target-SNARE (t-SNARE) interactions reduced syt stimulation of fusion. Using Sr2+ and Ba2+, we found that binding of syt to PS and t-SNAREs can be dissociated from activation of fusion, uncovering posteffector-binding functions for syt. Our data demonstrate that different syt isoforms are specialized to sense different ranges of divalent cations and that PS is an essential effector of Ca2+·syt action.

scholarly journals Analysis of the Protective Capacity of Three Streptococcus suis Proteins Induced under Divalent-Cation-Limited Conditions

2008 ◽  
Vol 76 (4) ◽  
pp. 1590-1598 ◽  
Author(s):  
Jesús Aranda ◽  
Maria Elena Garrido ◽  
Pilar Cortés ◽  
Montserrat Llagostera ◽  
Jordi Barbé
Keyword(s):  
Divalent Cation ◽  
Divalent Cations ◽  
Streptococcus Suis ◽  
Protein Product ◽  
Cation Binding ◽  
Target Sequence ◽  
Gene Promoters ◽  
Protective Capacity ◽  
Mobility Shift

ABSTRACT Streptococcus suis is a gram-positive pathogen that causes serious diseases in pigs and, in some cases, humans. Three genes of the virulent S. suis 89/1591 strain, encoding putative divalent-cation-binding lipoproteins, were isolated based on information obtained from the draft annotation files of this organism's genome. The products of these genes, which are inducible by divalent-cation deprivation, were subsequently purified, and their immunogenic and protective abilities were analyzed. All three proteins (SsuiDRAFT 0103, SsuiDRAFT 0174, and SsuiDRAFT 1237) were found to be immunogenic, but only one of them (SsuiDRAFT 0103) induced a significant protective response (87.5%, P = 0.01) against the same S. suis strain. Furthermore, the S. suis ssuiDRAFT 1240 gene (adcR), which encodes a predicted regulator of Zn2+ and/or Mn2+ uptake in streptococci, was cloned, and its protein product was purified. Electrophoretic mobility shift assays with purified S. suis AdcR protein showed experimentally, for the first time, that the AdcR DNA-binding sequence corresponds to the TTAACNRGTTAA motif. In addition, a requirement for either Zn2+ or Mn2+, but not Fe2+, to establish in vitro binding of AdcR to its target sequence and the ability of AdcR to bind the ssuiDRAFT 0103 and ssuiDRAFT 1237 gene promoters but not the promoter of the ssuiDRAFT 0174 gene were demonstrated. Taken together, these data suggest that SsuiDRAFT 0103 is a good candidate for vaccines against S. suis and support preliminary results indicating that bacterial envelope proteins involved in the uptake of divalent cations other than iron may be useful for protective purposes.

Early Platelet-Collagen Interactions in Whole Blood and Their Modifications by Aspirin and Dipyridamole Evaluated by a New Method (Basic Wave)

1985 ◽  
Vol 54 (04) ◽  
pp. 799-803 ◽  
Author(s):  
José Luís Pérez-Requejo ◽  
Justo Aznar ◽  
M Teresa Santos ◽  
Juana Vallés
Keyword(s):  
Whole Blood ◽  
Ex Vivo ◽  
Platelet Rich Plasma ◽  
White Blood Cells ◽  
Reversible Aggregation ◽  
Inhibitory Compounds ◽  
Rapid Generation ◽  
Stimulation Of ◽  
Collagen Stimulation

SummaryIt is shown that the supernatant of unstirred whole blood at 37° C, stimulated by 1 μg/ml of collagen for 10 sec, produces a rapid generation of pro and antiaggregatory compounds with a final proaggregatory activity which can be detected for more than 60 min on a platelet rich plasma (PRP) by turbidometric aggregometry. A reversible aggregation wave that we have called BASIC wave (for Blood Aggregation Stimulatory and Inhibitory Compounds) is recorded. The collagen stimulation of unstirred PRP produces a similar but smaller BASIC wave. BASIC’s intensity increases if erythrocytes are added to PRP but decreases if white blood cells are added instead. Aspirin abolishes “ex vivo” the ability of whole blood and PRP to generate BASIC waves and dipyridamole “in vitro” significantly reduces BASIC’s intensity in whole blood in every tested sample, but shows little effect in PRP.

DER EINFLUSS VON RESERPIN AUF DIE ANTITHYREOIDALE WIRKUNG VON KALIUMPERCHLORAT

1962 ◽  
Vol 39 (3) ◽  
pp. 423-430
Author(s):  
H. L. Krüskemper ◽  
F. J. Kessler ◽  
E. Steinkrüger
Keyword(s):  
Thyroid Gland ◽  
Male Rats ◽  
Normal Male ◽  
Tissue Respiration ◽  
List Type ◽  
Morphological Alterations ◽  
Hormone Synthesis ◽  
Stimulation Of

ABSTRACT 1. Reserpine does not inhibit the tissue respiration of liver in normal male rats (in vitro). 2. The decrease of tissue respiration of the liver with simultaneous morphological stimulation of the thyroid gland after long administration of reserpine is due to a minute inhibition of the hormone synthesis in the thyroid gland. 3. The morphological alterations of the thyroid in experimental hypothyroidism due to perchlorate can not be prevented with reserpine.

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