scholarly journals Measurement and characteristics of fusion of isolated membrane fractions from maize root tips

1980 ◽  
Vol 45 (1) ◽  
pp. 169-186
Author(s):  
E.A. Baydoun ◽  
D.H. Northcote
Keyword(s):  
Plasma Membrane ◽  
Membrane Fusion ◽  
Gel Electrophoresis ◽  
Divalent Cations ◽  
Polyacrylamide Gel Electrophoresis ◽  
Maize Root ◽  
Root Tips ◽  
Sulphydryl Group

Maize root tips were incubated in vivo with radioactive glucose, choline or diazotized sulphanilic acid. Membrane fractions were prepared from radioactive and non-radioactive roots. The transfer of radioactivity between mixed membrane fractions has enabled a quantitative system to be developed to study in vitro membrane fusion between Golgi apparatus and plasma membrane-rich fractions. Membrane fusion was found to be dependent on time, temperature, Mn2+ and Ca2+. Mn2+ was as effective as Ca2+, other divalent cations had a moderate or no effect. The effect of various substances, including inhibitors of microtubular and microfilament assembly and blocking reagents for sulphydryl group on membrane fusion has been investigated. The process appears to be dependent on the membrane proteins or glycoproteins; trypsinization of mixed membranes prior to the addition of Ca2+ inhibited significantly the fusion process. SDS-polyacrylamide gel electrophoresis of trypsin-treated membranes revealed the selective loss of one particular polypeptide which could play a role in membrane fusion.

In-vivo and in-vitro secretion of prolactin by lactating rat adenohypophyses as a function of intracellular age

1984 ◽  
Vol 101 (1) ◽  
pp. 27-32 ◽  
Author(s):  
F. Mena ◽  
G. Martínez-Escalera ◽  
C. Clapp ◽  
C. E. Grosvenor
Keyword(s):  
Pituitary Gland ◽  
Incubation Period ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyacrylamide Gel ◽  
Pituitary Glands ◽  
H 26

ABSTRACT Adenohypophysial prolactin of lactating rats was pulse-labelled by [3H]leucine injected i.v. at the time of removal of the pups. The [3H]prolactin concentration in the pituitary gland, analysed by polyacrylamide-gel electrophoresis, progressively fell as the time from labelling to removal of the pituitary gland increased from 8 to 24 h, which suggests that there was a loss of hormone as it aged within the gland. Suckling effectively provoked the depletion–transformation of total and [3H]prolactin (extracted at pH 7·2) when applied after 8 h but not when applied after either 16 or 24 h after removing the pups. In rats whose pups were removed for 8 h, suckling also depleted–transformed [3H]prolactin labelled 4 h, but not that labelled 1 h before suckling. The pituitary glands of other lactating rats were labelled with [3H]leucine injected i.v. at various times before removing the glands and incubating them in medium 199. The secretion into the medium of [3H]prolactin labelled either 4, 8, 16 or 24 h beforehand was maximal during the first 30 min then declined from 30 to 240 min of incubation. However, secretion of prolactin labelled 1 h and 10 min beforehand reached a maximum after 0·5–1 h and 2 h of incubation respectively, then remained constant during the remainder of the 4-h incubation period. The total 4-h secretion of [3H]prolactin was greatest (65% of preincubation concentration) from those glands labelled 4 h before in contrast to those labelled 10 min (15%) or 1 (38%), 8 (34%), 16 (18%) or 24 h (26%) before incubation. Taken together, these data suggest that prolactin synthesized 4 h earlier is more likely to be released in response to physiological stimuli than is more recently formed prolactin or prolactin which has remained in the pituitary gland for 16 h or longer. J. Endocr. (1984) 101, 27–32

Identification of D-Dimer-E Complex in Disseminated Intravascular Coagulation (DIC)

1979 ◽  
Author(s):  
A.N. Whitaker ◽  
E.A. Rowe ◽  
P.P. Masci ◽  
P.J. Gaffney
Keyword(s):  
Gel Electrophoresis ◽  
Degradation Products ◽  
Polyacrylamide Gel Electrophoresis ◽  
Stable Complex ◽  
Fibrin Degradation Products ◽  
Pneumococcal Sepsis ◽  
D Antigen ◽  
D Dimer

D-dimer (D2), a product of the plasmin lysis of cross-linked (XL) fibrin, but not of non-XL fibrin or fibrinogen, has been identified in the plasma of patients with DIC due to amniotic fluid embolism. In vitro, D is involved with fragment E as a stable complex (D2-E) but D2 -E has not been identified in vivo before. Fibrin degradation products (FDP) were studied in a patient having fulminant postsplenectomy pneumococcal sepsis and DIC, by immunoprecipitation with anti-fibrinogen (f) and anti-fragment E and characterization by SDS polyacrylamide gel electrophoresis (PAGE). With both antisera soluble HMW fibrin complexes, D2 and E but no X, Y or D were obtained from serum. D2 and E were identified in the supernatant after removing partially XL HMW complexes and fibrinogen from plasma with 2.5 M β-alanine. The presence D antigen in the D2-E complex precipitated by monospecific anti-E was confirmed by crossed Ag-Ab electrophoresis. Crossed Ag-Ab electrophoresis of serum in agarose gave E peaks of slow mobility and no fast-moving free E was found. Thus, D2-E complex exists in vivo and its easy identification, proving the lysis of XL fibrin, would be of value in studying thrombosis. D2-E complex has been identified in other patients with sepsis but at lower concentrations than described above.

Studies of the Metabolism of Asialotransferrins: Evidence that Transferrin Does not Undergo Desialylation in Vivo

1974 ◽  
Vol 46 (6) ◽  
pp. 763-774
Author(s):  
K.-L. Wong ◽  
P. A. Charlwood ◽  
M. W. C. Hatton ◽  
E. Regoeczi
Keyword(s):  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Biological Significance ◽  
Polyacrylamide Gel ◽  
Fractional Catabolic Rate ◽  
Sialic Acid Content ◽  
Catabolic Rate ◽  
Bovine Transferrin

1. Experiments are reported which aimed at determining whether transferrin loses sialyl residues from the carbohydrate side-chains during the biological lifetime of the molecule. To explore this possibility, transferrin fractions of relatively high sialic acid content (referred to as sialotransferrin) were prepared from purified rabbit and bovine transferrin by preparative polyacrylamide-gel electrophoresis. After labelling with 125I, the preparations were injected into a group of three rabbits each. From the plasma samples obtained between 1 h and 6–8 days after injection, transferrin was partially purified, mixed with 131I-labelled asialotransferrin of the corresponding species and run in preparative polyacrylamide-gel electrophoresis. In each specimen examined, the 125I radioactivity migrated ahead of the marker asialotransferrin, and no portion of the dose was detected with the electrophoretic mobility of asialotransferrin. 2. Evidence is presented that bovine transferrin desialylated in vitro remains detectable in the plasma of rabbits for intervals which are comparable with those found in previous studies with rabbit asialotransferrin. 3. A mathematical model is described for the computation of asialo- to sialotransferrin radioactivity ratios in the plasma, continuous desialylation of pulse-injected sialotransferrin being assumed. Calculations were made at various hypothetical rates of desialylation. 4. On the basis of the experimental data and the model it is concluded that transferrin (both rabbit and bovine) is not subjected to systematic desialylation in rabbits. Random desialylation of some transferrin could take place at rates less than 5% of the fractional catabolic rate of transferrin, which would be without any biological significance.

EFFECTS OF GONADOTROPHINS AND CYCLIC 3′,5′-AMP ON IN VITRO INCORPORATION OF [3H]URIDINE INTO RNA OF THE PREPUBERTAL RAT OVARY

1973 ◽  
Vol 72 (4) ◽  
pp. 771-785 ◽  
Author(s):  
Jan Jarlstedt ◽  
Lars Nilsson ◽  
Lars Hamberger ◽  
Kurt Ahrén
Keyword(s):  
Cyclic Nucleotide ◽  
Gel Electrophoresis ◽  
Incubation Medium ◽  
Polyacrylamide Gel Electrophoresis ◽  
Labelling Pattern ◽  
Total Rna ◽  
Rat Ovary ◽  
Prepubertal Rats

ABSTRACT In vivo and in vitro effects of FSH and LH on in vitro incorporation of [3H]uridine into RNA of the prepubertal rat ovary have been studied. RNA was fractionated with composite agarose-polyacrylamide gel electrophoresis. When FSH was injected into the prepubertal rats 4 h before incubation of the ovaries, the incorporation of labelled uridine into total RNA was decreased showing relatively more radioactivity concentrated to the RNA fractions lighter than 28S as compared to the controls. These effects were not seen when FSH was added to the incubation medium in vitro. When LH was added in vitro to the isolated ovaries a higher percentage of incorporated radioactivity was concentrated in the RNA fractions heavier than 28S without any change in the incorporation of [3H]uridine into total RNA. LH administered in vivo 30 min before incubation of the ovaries gave the same change in the labelling pattern between the RNA fractions as LH in vitro but in addition showed a decreased incorporation of radioactivity into total RNA. The in vitro effects of cyclic 3′,5′-AMP were also studied. When prepubertal rat ovaries were incubated in 10 mmol/l of this cyclic nucleotide, the incorporation of [3H] uridine into total RNA was decreased without any change in the labelling pattern.

scholarly journals Gel-electrophoretic identification of hen brain neurotoxic esterase, labelled with tritiated di-isopropyl phosphorofluoridate

1981 ◽  
Vol 199 (2) ◽  
pp. 323-333 ◽  
Author(s):  
D G Williams ◽  
M K Johnson
Keyword(s):  
Active Site ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
The Other ◽  
Particulate Fraction ◽  
Molecular Weights ◽  
The Brain ◽  
Neurotoxic Esterase

The particulate fraction from hen brain was labelled with [3H]di-isopropyl phosphorofluoridate (DiPF) and separated by polyacrylamide-gel electrophoresis. Four radioactive protein bands (1--4) of molecular weights 155000, 92000, 60000, and 30000 were resolved. Most of the labelling of bands 2, 3 and 4 was inhibited by preincubation with Paraoxon. The residue in band 4 was sensitive to pH 5.2. Successive treatments with Paraoxon and pH 5.2 resulted in the abolition of bands 3 and 4. Bands 1 and 2 contained one and two polypeptides respectively, whose labelling was sensitive to Mipafox, but one, in band 2, was sensitive to higher concentrations of Paraoxon. The concentrations of the other two polypeptides were 6.7 and 1.95 pmol of DiPF bound/g of brain in bands 1 and 2 respectively. Both were as sensitive to Mipafox as neurotoxic esterase and were also sensitive to phenyl benzylcarbamate. 4-Nitrophenyl di-n-pentylphosphinate given in vivo inhibited neurotoxic esterase and the labelling of the band-1 polypeptide by 82% and 84% respectively, but inhibited the labelling of the band 2 polypeptide by 51%. The phosphinate in vitro produced 98% inhibition of the labelling of the band-1 polypeptide, with only 26% inhibition of the band-2 polypeptide, under conditions sufficient to inhibit neurotoxic esterase totally. Both neurotoxic esterase and the band-1 polypeptide were found in the forebrain at 1.74-fold their concentration in the rest of the brain, whereas the band-2 polypeptide was uniformly distributed. The evidence indicates that the Mipafox-sensitive polypeptide in band 1 is the [3H]DiPF-labelled active-site subunit of neurotoxic esterase. The catalytic-centre activity of the enzyme for phenyl valerate hydrolysis was found to be 2.6 x 10(5) min-1.

scholarly journals The Polybasic Juxtamembrane Region of Sso1p Is Required for SNARE Function In Vivo

2005 ◽  
Vol 4 (12) ◽  
pp. 2017-2028 ◽  
Author(s):  
Jeffrey S. Van Komen ◽  
Xiaoyang Bai ◽  
Travis L. Rodkey ◽  
Johanna Schaub ◽  
James A. McNew
Keyword(s):  
Plasma Membrane ◽  
Membrane Fusion ◽  
Transmembrane Domain ◽  
Transmembrane Helix ◽  
Specific Interaction ◽  
Coiled Coil ◽  
Snare Complex ◽  
Juxtamembrane Region

ABSTRACT Exocytosis in Saccharomyces cerevisiae requires the specific interaction between the plasma membrane t-SNARE complex (Sso1/2p;Sec9p)and a vesicular v-SNARE (Snc1/2p). While SNARE proteins drive membrane fusion, many aspects of SNARE assembly and regulation are ill defined. Plasma membrane syntaxin homologs (including Sso1p) contain a highly charged juxtamembrane region between the transmembrane helix and the“ SNARE domain” or core complex domain. We examined this region in vitro and in vivo by targeted sequence modification, including insertions and replacements. These modified Sso1 proteins were expressed as the sole copy of Sso in S. cerevisiae and examined for viability. We found that mutant Sso1 proteins with insertions or duplications show limited function, whereas replacement of as few as three amino acids preceding the transmembrane domain resulted in a nonfunctional SNARE in vivo. Viability is also maintained when two proline residues are inserted in the juxtamembrane of Sso1p, suggesting that helical continuity between the transmembrane domain and the core coiled-coil domain is not absolutely required. Analysis of these mutations in vitro utilizing a reconstituted fusion assay illustrates that the mutant Sso1 proteins are only moderately impaired in fusion. These results suggest that the sequence of the juxtamembrane region of Sso1p is vital for function in vivo, independent of the ability of these proteins to direct membrane fusion.

scholarly journals Complex Formation of Fibrin Monomers Studied by Gel Filtration and Polyacrylamide Gel Electrophoresis

1977 ◽  
Author(s):  
R. Hafter ◽  
M. Baumgärtner ◽  
R. v.Hugo ◽  
H. Graeff
Keyword(s):  
Complex Formation ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Molecular Structures ◽  
Molar Ratio ◽  
In Vitro Tests ◽  
Fibrin Monomers

Estimation and characterization of thrombin mediated products of fibrinogen (soluble fibrin monomer complexes=SFMC) can be achieved by gel filtration and PAA-gel electrophoresis. In order to gain further information about complex formation in vitro tests were performed. Fibrin monomers were prepared from fibrin dissolved in 3 M KBr after action of thrombin (des-AB-fibrin) and reptilase (des-A-fibrin) on fibrinogen and added to plasma. In a certain range of fibrin concentration (up to 3% for des-AB-fibrin and up to 15% for des-A-fibrin) soluble complexes are formed with fibrinogen in a 1:1 molar ratio. Further increase of fibrin percentage results in a partial precipitation of complexes. Additionally, a marked increase of SFMC (up to 60 %) is observed with des-A-fibrin. This latter reaction indicates complex formation with a higher ratio of fibrinogen involved. An influence of temperature on complex forming could be observed. Gel filtration at 37°C reveals a shift in the elu-tion profile, indicating dissociation of high molecular weight complexes (5 million daltons) to lower molecular structures. The dissociation behaviour of SFMC from plasma samples of patients with hypercoagulability is similar. However, crosslinked fibrinoligomers showed no temperature dependant dissociation behaviour. It is concluded that SFMC exist during the state of hypercoagulability in vivo predominantly as a dimeric fibrin-fibrinogen complex.

Heat shock of Drosophila melanogaster induces the synthesis of new messenger RNAs and proteins

1978 ◽  
Vol 283 (997) ◽  
pp. 391-406 ◽  
Keyword(s):  
Heat Shock ◽  
Heat Shock Proteins ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Messenger Rnas ◽  
Sodium Dodecylsulphate ◽  
Shock Proteins ◽  
Kinetics Of

The heat shock proteins, labelled in vivo with [ 35 S]methionine, were separated by sodium dodecylsulphate-polyacrylamide gel electrophoresis and fingerprinted after tryptic digestion. Eight distinct heat shock polypeptides were characterized in this way. Heat shock messenger RNAs were isolated and partially purified. Assayed in vitro for protein synthesis, they were found to code for heat shock polypeptides. Some parameters of the kinetics of in vivo synthesis of the heat shock proteins are presented.

scholarly journals The extraction from maize (Zea mays) root cells of membrane-bound protein with Ca2+-dependent ATPase activity and its possible role in membrane fusion in vitro

1981 ◽  
Vol 193 (3) ◽  
pp. 781-792 ◽  
Author(s):  
E A H Baydoun ◽  
D H Northcote
Keyword(s):  
Zea Mays ◽  
Atpase Activity ◽  
Membrane Fusion ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Sodium Deoxycholate ◽  
Root Cells ◽  
Dependent Atpase

Membrane fusion in vitro between Golgi apparatus- and plasma-membrane-rich fractions isolated from maize (Zea mays) roots was found to be dependent on Ca2+ and the membrane proteins. Trypsin treatment of mixed membrane fractions before the addition of Ca2+ inhibited their ability to fuse. It resulted also in a selective and progressive elimination of a characteristic intense polypeptide band (B1) on gel electrophoresis. This polypeptide was not removed by chymotrypsin or thermolysin. B1 is an integral membrane protein with an exposed portion to the outside. Sodium deoxycholate was used to solubilize the proteins of mixed membrane fractions. Extracted proteins analysed by non-SDS (sodium dodecyl sulphate) polyacrylamide-gel electrophoresis revealed the presence of four isolated bands. When re-electrophoresed in the presence of SDS, one of these bands exhibited the same mobility as polypeptide B1. Enzymic staining of non-SDS-polyacrylamide gels showed that this protein has Ca2+- and Mg2+-dependent ATPase activity. Its possible role in membrane fusion is discussed.

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