scholarly journals Soluble C3 proconvertase and convertase of the classical pathway of human complement. Conditions of stabilization in vitro

1985 ◽  
Vol 226 (2) ◽  
pp. 429-436 ◽  
Author(s):  
M B Villiers ◽  
N M Thielens ◽  
M G Colomb
Keyword(s):  
Density Gradient ◽  
Binding Protein ◽  
Sucrose Density Gradient ◽  
Density Gradient Ultracentrifugation ◽  
Classical Pathway ◽  
Human Complement ◽  
Proteolytic Fragment ◽  
The Individual

Soluble classical-pathway C3 convertase and proconvertase were prepared from purified C4b-C2ox complex in the presence of Ni2+; the two complexes, stable for at least 15 h at 4 degrees C, were isolated by sucrose-density-gradient ultracentrifugation. The C3 convertase alone was able to cleave C3, and its decay was accelerated in the presence of C4-binding protein. The individual roles of Ni2+ and I2 treatment of C2 in the stabilization of the complexes seemed to be different and additive. 63Ni2+ binding coupled to h.p.l.c. analysis showed that 63Ni2+ bound only to the C2ox proteolytic fragment a (1 mol/mol) with a Kd of 26 microM. Competition studies between Ni2+ and Mg2+ indicated that only half of the Ni2+ bound to the C3 convertase was removed by Mg2+, whereas, in the same conditions, Ni2+ bound to C2ox proteolytic fragment a was not displaced, suggesting the presence of two sets of sites on the convertase. EDTA prevented the formation of both C3 convertase and proconvertase; EDTA had no effect on the preformed C3 convertase, whereas it dissociated the preformed proconvertase.

The Binding of Lithium Carmine to a2-Macroglobulin

1969 ◽  
Vol 24 (11) ◽  
pp. 1442-1447 ◽  
Author(s):  
J. J. Picard ◽  
J. F. Heremans
Keyword(s):  
Human Serum ◽  
Density Gradient ◽  
Protein Complex ◽  
Gel Filtration ◽  
Heavy Fraction ◽  
Normal Human Serum ◽  
Density Gradient Ultracentrifugation ◽  
Γ Globulins ◽  
Normal Human

The colloidal dye lithium carmine was added in vitro to normal human serum. Electrophoretic experiments showed that the dye was associated mainly with α2-globulins, small amounts with the albumin and only traces with the γ-globulins. The main complex was eluted with the macroglobulin peak obtained by gel filtration on Sephadex G-200 and sedimented in the heavy fraction on density gradient ultracentrifugation. The dye-protein complex could be precipitated with an antiserum specific for a2-macroglobulin. Gel filtration of a solution of pure a2-macroglobulin, to which lithium carmine was added, demonstrated that the dye was bound to this protein.

scholarly journals Identification of storage-protein messenger RNA of the fleshfly Sarcophaga peregrina

1982 ◽  
Vol 203 (3) ◽  
pp. 571-575 ◽  
Author(s):  
T Tahara ◽  
Y Maeda ◽  
A Kuroiwa ◽  
K Ueno ◽  
M Obinata ◽  
...  
Keyword(s):  
Density Gradient ◽  
Storage Protein ◽  
Messenger Rna ◽  
Fat Body ◽  
Density Gradient Centrifugation ◽  
Signal Sequence ◽  
Gradient Centrifugation ◽  
Sucrose Density Gradient ◽  
Sucrose Density Gradient Centrifugation

Storage-protein mRNA was found to be abundant in poly(A)-containing RNA extracted from the fat-body of third-instar larvae of Sarcophaga peregrina (fleshfly). This RNA sedimented at the position of 19S on sucrose-density-gradient centrifugation and the product of its translation in vitro was 75K protein (protein of mol.wt. 75 000), which was precipitated specifically with antibody against storage protein. This product was suggested to contain a signal sequence that is missing in mature storage protein. The poly(A)-containing RNA was also found to contain much of another mRNA coding for 25K protein (protein of mol.wt. 25 000), but the function of this protein is unknown.

scholarly journals The effect of phenobarbital on the submicrosomal distribution of uridine diphosphate glucuronyltransferase from rat liver

1970 ◽  
Vol 117 (2) ◽  
pp. 319-324 ◽  
Author(s):  
G. J. Mulder
Keyword(s):  
Enzyme Activity ◽  
Density Gradient ◽  
Rat Liver ◽  
Microsomal Fraction ◽  
Specific Activity ◽  
Sucrose Density Gradient ◽  
Male Rats ◽  
Uridine Diphosphate ◽  
Triton X

1. The detergent Triton X-100 activates UDP glucuronyltransferase from rat liver in vitro six- to seven-fold with p-nitrophenol as substrate. The enzyme activity when measured in the presence of Triton X-100 is increased significantly by pretreatment of male rats with phenobarbital for 4 days (90mg/kg each day intraperitoneally). If no Triton X-100 is applied in vitro such an increase could not be shown. In all further experiments the enzyme activity was measured after activation by Triton X-100. 2. The Km of the enzyme for the substrate p-nitrophenol does not change on phenobarbital pretreatment. 3. When the microsomal fraction from the liver of untreated rats is subfractionated on a sucrose density gradient, 47% of the enzyme activity is recovered in the rough-surfaced microsomal fraction, which also has a higher specific activity than the smooth-surfaced fraction. 4. Of the increase in activity after the phenobarbital pretreatment 50% occurs in the smooth-surfaced fraction, 19% in the rough-surfaced fraction and 31% in the fraction located between the smooth- and rough-surfaced microsomal fractions on the sucrose density gradient. 5. The latency of the enzyme in vitro, as shown by the effect of the detergent Triton X-100, is discussed in relation to the proposed heterogeneity of UDP glucuronyltransferase.

scholarly journals THE SEDIMENTATION PROPERTIES OF THE SKIN-SENSITIZING ANTIBODIES OF RAGWEED-SENSITIVE PATIENTS

1964 ◽  
Vol 120 (1) ◽  
pp. 31-43 ◽  
Author(s):  
Burton R. Andersen ◽  
Wilton E. Vannier
Keyword(s):  
Density Gradient ◽  
Sedimentation Rate ◽  
Gradient Method ◽  
Sucrose Density Gradient ◽  
Major Portion ◽  
Density Gradient Ultracentrifugation ◽  
Cell Method ◽  
Average Value ◽  
Γ Globulins

The sedimentation coefficients of the skin-sensitizing antibodies to ragweed were evaluated by the moving partition cell method and the sucrose density gradient method. The most reliable results were obtained by sucrose density gradient ultracentrifugation which showed that the major portion of skin-sensitizing antibodies to ragweed sediment with an average value of 7.7S (7.4 to 7.9S). This is about one S unit faster than γ-globulins (6.8S). The data from the moving partition cell method are in agreement with these results. Our studies failed to demonstrate heterogeneity of the skin-sensitizing antibodies with regard to sedimentation rate.

scholarly journals The IGF system in neuroblastoma xenografts: focus on IGF-binding protein-6

2002 ◽  
Vol 172 (3) ◽  
pp. 467-476 ◽  
Author(s):  
P Grellier ◽  
D Berrebi ◽  
M Peuchmaur ◽  
S Babajko
Keyword(s):  
Binding Protein ◽  
Neuroblastoma Cell ◽  
Homogeneous Distribution ◽  
Tunel Staining ◽  
Human Neuroblastoma ◽  
Proteolytic Fragment ◽  
Igf Binding ◽  
Igf Binding Protein

With a view to investigating the implication of IGF-binding protein-6 (IGFBP-6) in the growth of neuroblastomas, nude mice were injected with IGFBP-6-expressing or control IGR-N-91 human neuroblastoma cells and the resulting xenografts examined. Expression of IGFBP-3, IGFBP-4 and type 1 and type 2 IGF receptor messengers was similar in control tumours and equal-sized IGFBP-6-expressing tumours that had developed. IGF-II was more strongly expressed in control tumours, and IGFBP-6-expressing tumours contained less IGFBP-2 than controls. In both populations, there was a significant positive correlation between IGF-II and IGFBP-2 expression. In small IGFBP-6-expressing xenografts where tumour development had apparently been arrested, haematoxylin--eosin and TUNEL staining revealed numerous apoptotic cells. In situ hybridization indicated homogeneous distribution of the IGFBP-6 signal in test tumours. In cell culture, IGFBP-6-expressing cells expressed similar amounts of IGFBP-2, IGF-II and N-myc mRNAs as control cells; but media conditioned by IGFBP-6-expressing cells contained less intact IGFBP-2 protein, with no increase in its proteolytic fragment. In media treated with plasminogen, in which IGFBP-2 was proteolysed, IGFBP-6 was increased. With its especially strong affinity for IGF-II and its resistance to proteolysis, IGFBP-6 would act by sequestering IGF-II, hence inhibiting its mitogenic and anti-apoptotic effects. In excess, IGFBP-6 would displace IGF-II from IGFBP-2 whose potentiation of IGF-II action would cease and whose susceptibility to degradation would be increased. This study therefore shows that IGFBP-6 plays a role in neuroblastoma cell growth in vivo and in vitro and that stable overexpression of IGFBP-6 leads to alteration of the initial balance between the IGFBPs.

scholarly journals Subcellular localization of ferritin and iron taken up by rat hepatocytes

1989 ◽  
Vol 262 (2) ◽  
pp. 685-688 ◽  
Author(s):  
J C Sibille ◽  
M Ciriolo ◽  
H Kondo ◽  
R R Crichton ◽  
P Aisen
Keyword(s):  
Acid Phosphatase ◽  
Cytochrome C ◽  
Subcellular Localization ◽  
Cytochrome C Oxidase ◽  
Density Gradient ◽  
Rat Hepatocytes ◽  
Sucrose Density Gradient ◽  
Gel Chromatography ◽  
Density Gradient Ultracentrifugation

The subcellular localization of ferritin and its iron taken up by rat hepatocytes was investigated by sucrose-density-gradient ultracentrifugation of cell homogenates. After incubation of hepatocytes with 125I-labelled [59Fe]ferritin, cells incorporate most of the labels into structures equilibrating at densities where acid phosphatase and cytochrome c oxidase are found, suggesting association of ferritin and its iron with lysosomes or mitochondria. Specific solubilization of lysosomes by digitonin treatment indicates that, after 8 h incubation, most of the 125I is recovered in lysosomes, whereas 59Fe is found in mitochondria as well as in lysosomes. As evidenced by gel chromatography of supernatant fractions, 59Fe accumulates with time in cytosolic ferritin. To account for these results a model is proposed in which ferritin, after being endocytosed by hepatocytes, is degraded in lysosomes, and its iron is released and re-incorporated into cytosolic ferritin and, to a lesser extent, into mitochondria.

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