scholarly journals Transient membrane association of the precursors of cathepsin C during their transfer into lysosomes

1991 ◽  
Vol 275 (3) ◽  
pp. 797-800 ◽  
Author(s):  
V Burge ◽  
F Mainferme ◽  
R Wattiaux
Keyword(s):  
Endoplasmic Reticulum ◽  
Lysosomal Enzyme ◽  
Osmotic Shock ◽  
Subcellular Fractions ◽  
Membrane Association ◽  
Cathepsin C ◽  
Sds Page ◽  
Morris Hepatoma ◽  
Triton X

Transport of the lysosomal enzyme cathepsin C was studied in Morris hepatoma 7777 cells. Subcellular fractions obtained after isopyenic centrifugation in sucrose gradients of labelled cell homogenates were sequentially extracted by hypo-osmotic shock, Na2CO3 and Triton X-100. Polypeptides related to cathepsin C were immunoprecipitated and analysed by SDS/PAGE and fluorography. At early times after synthesis and for up to 60 min, precursor polypeptides of cathepsin C are distributed in endoplasmic reticulum and Golgi fractions, in membrane-associated form, as Triton X-100 is necessary for their extraction. At 2 h and later after synthesis, intermediate and mature forms of the enzyme can be totally extracted by hypo-osmotic shock from gradient fractions corresponding to the lysosomes of Morris hepatoma 7777 cells.

scholarly journals Quantification of apolipoprotein B-48 and B-100 in rat liver endoplasmic reticulum and Golgi fractions

1992 ◽  
Vol 285 (1) ◽  
pp. 153-159 ◽  
Author(s):  
I J Cartwright ◽  
J A Higgins
Keyword(s):  
Endoplasmic Reticulum ◽  
Rat Liver ◽  
Smooth Endoplasmic Reticulum ◽  
Protein A ◽  
Subcellular Fractions ◽  
Apo B ◽  
Sds Page ◽  
Membrane Bound ◽  
Triton X ◽  
Quantitative Immunoblotting

We have developed a method for measurement of apolipoprotein (apo) B-48 and apo B-100 in blood and subcellular fractions of rat liver based on SDS/PAGE followed by quantitative immunoblotting using 125I-Protein A. Standard curves were prepared in each assay using apo B prepared from total rat lipoproteins by extraction with tetramethylurea. Subcellular fractions (rough and smooth endoplasmic reticulum and Golgi fractions) were prepared from rat liver and separated into membrane and cisternal-content fractions. For quantification, membrane fractions were solubilized in Triton X-100, and the apo B was immunoprecipitated before separation by SDS/PAGE and immunoblotting. Content fractions were concentrated by ultrafiltration and separated by SDS/PAGE without immunoprecipitation. Quantification of apo B in subcellular fractions and detection of apo B by immunoblotting yielded consistent results. In all fractions apo B-48 was the major form, accounting for approximately three-quarters of the total apo B. By using marker enzymes as internal standards, it was calculated that all of the apo B was recovered in the endoplasmic reticulum and Golgi fractions, with approximately 80% of each form of apo B in the endoplasmic reticulum. More than 90% of the apo B of the rough- and smooth-endoplasmic-reticulum fractions was membrane-bound, whereas approx. 33 and 15% of the apo B of the cis-enriched Golgi fractions and trans-enriched Golgi fractions respectively were membrane-bound.

scholarly journals Mannosyl carrier functions of retinyl phosphate and dolichyl phosphate in rat liver endoplasmic reticulum

1983 ◽  
Vol 210 (2) ◽  
pp. 541-547 ◽  
Author(s):  
K E Creek ◽  
D J Morré ◽  
C S Silverman-Jones ◽  
Y Shidoji ◽  
L M De Luca
Keyword(s):  
Endoplasmic Reticulum ◽  
Golgi Apparatus ◽  
Rat Liver ◽  
Subcellular Fractions ◽  
Transfer Activity ◽  
Dolichyl Phosphate ◽  
Endoplasmic Reticulum Protein ◽  
Triton X

Of the subcellular fractions of rat liver the endoplasmic reticulum was the most active in GDP-mannose: retinyl phosphate mannosyl-transfer activity. The synthesis of retinyl phosphate mannose reached a maximum at 20-30 min of incubation and declined at later times. Retinyl phosphate mannose and dolichyl phosphate mannose from endogenous retinyl phosphate and dolichyl phosphate could also be assayed in the endoplasmic reticulum. About 1.8 ng (5 pmol) of endogenous retinyl phosphate was mannosylated per mg of endoplasmic reticulum protein (15 min at 37 degrees C, in the presence of 5 mM-MnCl2), and about 0.15 ng (0.41 pmol) of endogenous retinyl phosphate was mannosylated with Golgi-apparatus membranes. About 20 ng (13.4 pmol) of endogenous dolichyl phosphate was mannosylated in endoplasmic reticulum and 4.5 ng (3 pmol) in Golgi apparatus under these conditions. Endoplasmic reticulum, but not Golgi-apparatus membranes, catalysed significant transfer of [14C]mannose to endogenous acceptor proteins in the presence of exogenous retinyl phosphate. Mannosylation of endogenous acceptors in the presence of exogenous dolichyl phosphate required the presence of Triton X-100 and could not be detected when dolichyl phosphate was solubilized in liposomes. Dolichyl phosphate mainly stimulated the incorporation of mannose into the lipid-oligosaccharide-containing fraction, whereas retinyl phosphate transferred mannose directly to protein.

scholarly journals The diglyceride kinase of rat cerebral cortex

1971 ◽  
Vol 122 (2) ◽  
pp. 171-179 ◽  
Author(s):  
E. G. Lapetina ◽  
J. N. Hawthorne
Keyword(s):  
Cerebral Cortex ◽  
Phosphatidic Acid ◽  
Nerve Ending ◽  
Osmotic Shock ◽  
Sodium Deoxycholate ◽  
Acid Synthesis ◽  
Optimum Concentration ◽  
Subcellular Fractions ◽  
Rat Cerebral Cortex ◽  
Triton X

1. Formation of phosphatidic acid by diglyceride kinase (EC 2.7.1.-) in the presence of ATP and Mg2+ was shown in a homogenate and subcellular fractions of rat cerebral cortex. 2. The kinase was activated by Mg2+. Ca2+ activated to a smaller extent but was inhibitory in the presence of optimum concentration of Mg2+. Activity was greatly increased in the presence of added 1,2-diglyceride. 3. Sodium deoxycholate markedly stimulated the reaction, but other detergents (Cutscum and Triton X-100) did not. 4. Diglyceride kinase was concentrated in the supernatant and microsomal fractions from rat cerebral cortex. The distribution of the kinase in the particulate fractions resembled that of acetylcholinesterase and 5′-nucleotidase. 5. The rate of phosphatidic acid synthesis by the diglyceride kinase route was much greater than reported rates for acylation of 3-glycerophosphate and was also very rapid in comparison with the rates of other steps in the synthesis of phosphoinositides. 6. Acetylcholine had no stimulatory effect on diglyceride kinase of isolated intact nerve-ending particles or of nerve-ending membranes obtained after osmotic shock.

scholarly journals Calpain activity in patients with thrombotic thrombocytopenic purpura is associated with platelet microparticles

Blood ◽  
1992 ◽  
Vol 80 (9) ◽  
pp. 2246-2251 ◽  
Author(s):  
JG Kelton ◽  
TE Warkentin ◽  
CP Hayward ◽  
WG Murphy ◽  
JC Moore
Keyword(s):  
Thrombotic Thrombocytopenic Purpura ◽  
Membrane Association ◽  
Membrane Glycoproteins ◽  
Calpain Activity ◽  
Thrombocytopenic Purpura ◽  
Calcium Dependent ◽  
Active Protease ◽  
Triton X ◽  
Platelet Microparticles

Abstract Thrombotic thrombocytopenic purpura (TTP) is characterized by thrombocytopenia and disseminated platelet thrombi throughout the microvasculature. Studies by our group have demonstrated calcium- dependent proteolytic activity (calpain) that is no longer detectable in the serum of patients with acute TTP after their recovery. The purpose of this study was to investigate if the protease activity of TTP was detectable in plasma and, therefore, not an in vitro phenomenon secondary to the formation of serum. Additionally, we looked for evidence of membrane association of the active protease in the patients' samples, which would explain the persistence of its activity in the presence of plasma inhibitors. Acute TTP samples, both serum and plasma, were collected from 10 patients with TTP. Calpain was measured using bioassays for enzyme activity and also by detection of the protein using immunoblotting with an anticalpain monoclonal antibody (MoAb). In all instances, calpain could be detected both functionally and antigenically in the acute TTP sera and plasma. No calpain activity could be detected in any of the controls, although antigenic calpain was detectable in one sample from a patient who had undergone cardiopulmonary bypass surgery. To investigate whether the calpain was associated with microparticles in the plasma, the TTP plasma samples were ultrafiltered and ultracentrifuged. Activity was not lost by passage across a 0.2-micron filter but was detectable only in the pellet following ultracentrifugation. Membrane association of the calpain in the microparticles also was demonstrated using solubilization with Triton X-100. Immunoprecipitation studies demonstrated that the calpain activity could be removed by MoAbs against platelet membrane glycoproteins (IX and IIb/IIa) but not by a MoAb against red blood cell membrane glycophorin. These studies indicate that active calpain is associated with platelet microparticles in plasma from patients with TTP.

scholarly journals Impact of Extraction Method on the Detection of Quality Biomarkers in Normal vs. DFD Meat

Foods ◽  
2021 ◽  
Vol 10 (5) ◽  
pp. 1097
Author(s):  
Laura González-Blanco ◽  
Yolanda Diñeiro ◽  
Andrea Díaz-Luis ◽  
Ana Coto-Montes ◽  
Mamen Oliván ◽  
...  
Keyword(s):  
Extraction Method ◽  
Protein Pattern ◽  
Stress Protein ◽  
Electrophoretic Pattern ◽  
Extraction Methods ◽  
Sds Page ◽  
Weight Protein ◽  
Triton X ◽  
Protein Extractability ◽  
Selection Of

The objective of this work was to demonstrate how the extraction method affects the reliability of biomarker detection and how this detection depends on the biomarker location within the cell compartment. Different extraction methods were used to study the sarcoplasmic and myofibrillar fractions of the Longissimus thoracis et lumborum muscle of young bulls of the Asturiana de los Valles breed in two quality grades, standard (Control) or dark, firm, and dry (DFD) meat. Protein extractability and the expression of some of the main meat quality biomarkers—oxidative status (lipoperoxidation (LPO) and catalase activity (CAT)), proteome (SDS-PAGE electrophoretic pattern), and cell stress protein (Hsp70)—were analyzed. In the sarcoplasmic fraction, buffers containing Triton X-100 showed significantly higher protein extractability, LPO, and higher intensity of high-molecular-weight protein bands, whereas the TES buffer was more sensitive to distinguishing differences in the protein pattern between the Control and DFD meat. In the myofibrillar fraction, samples extracted with the lysis buffer showed significantly higher protein extractability, whereas samples extracted with the non-denaturing buffer showed higher results for LPO, CAT, and Hsp70, and higher-intensity bands in the electrophoretic pattern. These findings highlight the need for the careful selection of the extraction method used to analyze the different biomarkers considering their cellular location to adapt the extractive process.

A metalloproteinase extracellularly released byCrithidia deanei

2003 ◽  
Vol 49 (10) ◽  
pp. 625-632 ◽  
Author(s):  
Claudia Masini d'Avila-Levy ◽  
Rodrigo F Souza ◽  
Rosana C Gomes ◽  
Alane B Vermelho ◽  
Marta H Branquinha
Keyword(s):  
Molecular Mass ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Residual Activity ◽  
Major Protein ◽  
Motile Cells ◽  
Sds Page ◽  
Triton X

Actively motile cells from a cured strain of Crithidia deanei released proteins in phosphate buffer (pH 7.4). The molecular mass of the released polypeptides, which included some proteinases, ranged from 19 to 116 kDa. One of the major protein bands was purified to homogeneity by a combination of anion-exchange and gel filtration chromatographs. The apparent molecular mass of this protein was estimated to be 62 kDa by sodium dodecyl sulfate – polyacrylamide gel electrophoresis (SDS–PAGE). The incorporation of gelatin into SDS–PAGE showed that the purified protein presented proteolytic activity in a position corresponding to a molecular mass of 60 kDa. The enzyme was optimally active at 37 °C and pH 6.0 and showed 25% of residual activity at 28 °C for 30 min. The proteinase was inhibited by 1,10-phenanthroline and EDTA, showing that it belonged to the metalloproteinase class. A polyclonal antibody to the leishmanial gp63 reacted strongly with the released C. deanei protease. After Triton X-114 extraction, an enzyme similar to the purified metalloproteinase was detected in aqueous and detergent-rich phases. The detection of an extracellular metalloproteinase produced by C. deanei and some other Crithidia species suggests a potential role of this released enzyme in substrate degradation that may be relevant to the survival of trypanosomatids in the host.Key words: endosymbiont, trypanosomatid, extracellular, proteinase.

CONTROL OF CELLULAR CONTRACTION BY CALCIUM IN VORTICELLA

1994 ◽  
Vol 189 (1) ◽  
pp. 163-177 ◽  
Author(s):  
K Katoh ◽  
Y Naitoh
Keyword(s):  
Endoplasmic Reticulum ◽  
Cell Body ◽  
Ruthenium Red ◽  
Measurable Effect ◽  
External Application ◽  
Sustained Contraction ◽  
Intracellular Injection ◽  
Contractile System ◽  
Triton X

1. Vorticella extracted with Triton X-100 contracted (i.e. the cell body shrank and the stalk coiled) when the external Ca2+ concentration was raised. The degree of contraction increased with increasing Ca2+ concentration. 2. The threshold Ca2+ concentration for shrinkage of the cell body was identical with that for coiling of the stalk in Vorticella extracted with Triton X-100. 3. Living Vorticella showed a graded shrinkage of the cell body when Ca2+ buffer was injected into the cell body, while the stalk showed coiling of an all-or-nothing type. The degree of shrinkage of the cell body increased with increasing free Ca2+ concentration of the buffer. 4. Living Vorticella showed a sustained contraction in response to external application or intracellular injection of caffeine. The effect of caffeine was inhibited by intracellular injection of procaine or Ruthenium Red. 5. Vorticella injected with Ruthenium Red showed graded shrinkage of the cell body as well as graded coiling of the stalk when Ca2+ buffer was injected into the cell body. 6. Caffeine, procaine and Ruthenium Red had no measurable effect on Ca2+-activated contraction in Vorticella extracted with Triton X-100. 7. It is assumed that regenerative liberation of Ca2+ from the endoplasmic reticulum and/or membranous tubules in the contractile system (Ca2+-induced Ca2+ release) is responsible for evoking contraction of an all-or-nothing type following stimulation in living Vorticella.

scholarly journals Cultured human umbilical vein endothelial cells contain a membrane glycoprotein immunologically related to platelet glycoprotein Ib

Blood ◽  
1988 ◽  
Vol 71 (1) ◽  
pp. 234-237 ◽  
Author(s):  
JD Sprandio ◽  
SS Shapiro ◽  
P Thiagarajan ◽  
S McCord
Keyword(s):  
Endothelial Cells ◽  
Umbilical Vein ◽  
Sodium Dodecyl ◽  
Platelet Glycoprotein ◽  
Glycoprotein Ib ◽  
Single Class ◽  
Human Umbilical Vein ◽  
Sds Page ◽  
Umbilical Vein Endothelial Cells ◽  
Triton X

Using a platelet glycoprotein Ib (GpIb)-specific monoclonal antibody, AP-1, we have studied cultured human umbilical vein endothelial cells (HUVEC) for the presence of GpIb. Radiolabeled AP-1 bound specifically and saturably to HUVEC in suspension and detected a single class of binding sites (100,000/cell). When Triton X-100 extracts of HUVEC were chromatographed on wheat germ agglutinin (WGA)-Sepharose, radioiodinated, precipitated with AP-1, and subjected to reduced sodium dodecyl sulfate-polyacrylamide electrophoresis (SDS-PAGE), major radioactive bands of 228,000, 145,000, and 130,000 were seen. The latter two bands correspond to the 156,000 and 140,000 bands, representing GpIb alpha and glycocalicin, respectively, which are seen when platelets are subjected to the same procedure. The 228,000 band corresponds to a band previously noted in immunoprecipitates of platelet GpIb but not fully explained. When HUVEC were grown in the presence of 35S-methionine, extracted with Triton X-100, chromatographed on WGA-Sepharose, immunoprecipitated with AP-1, and subjected to reduced SDS-PAGE, radioactive bands of 210,000, 156,000, and 90,000 were seen. We conclude that cultured HUVEC synthesize and express on their surface a glycoprotein immunologically related to platelet GpIb.

scholarly journals SDS/PAGE characteristics of protein kinases tightly associated with chick embryo brain ribosomes.

1999 ◽  
Vol 46 (4) ◽  
pp. 911-917
Author(s):  
M Sanecka-Obacz
Keyword(s):  
Chick Embryo ◽  
Protein Kinases ◽  
Embryo Development ◽  
Sds Page ◽  
Casein Kinases ◽  
Triton X ◽  
Brain Ribosomes ◽  
Embryo Brain ◽  
Exogenous Substrates

Protein kinases tightly associated with chick embryo brain ribosomes washed with Triton X-100 and KCl were characterized by their ability to phosphorylate ribosomes and two exogenous substrates, histone IIA and casein. c-AMP-dependent kinase (PKA) and casein kinases (CK1, CK2) were examined in the presence of specific modulators by SDS/PAGE followed by renaturation in gel assay according to Kameshita & Fujisawa (Anal. Biochem. 1989, 183, 139-143). Basing on these data it can be presumed that PKA activity increases, but the levels of CK2 and CK1 decrease during chick embryo development.

scholarly journals Evidence for the involvement of a 66 kDa membrane protein in the synthesis of sterolglucoside in Saccharomyces cerevisiae.

1995 ◽  
Vol 42 (2) ◽  
pp. 269-274 ◽  
Author(s):  
U Lenart ◽  
J Haplova ◽  
P Magdolen ◽  
V Farkas ◽  
G Palamarczyk
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Molecular Mass ◽  
Deae Cellulose ◽  
Yeast Saccharomyces Cerevisiae ◽  
Sds Page ◽  
Nonionic Detergent ◽  
Membrane Bound ◽  
Labeled Probe ◽  
Triton X ◽  
Cellulose Column

The membrane-bound sterolglucoside synthase from the yeast Saccharomyces cerevisiae has been solubilized by nonionic detergent, Nonidet P-40, Triton X-100, and partially purified by DEAE-cellulose column chromatography and ammonium sulfate fractionation. SDS/PAGE of the purified fraction revealed the presence of two protein bands of molecular mass 66 kDa and 54 kDa. In an attempt to identify further the polypeptide chain of sterolglucoside synthase, the partially purified enzyme was treated with [di-125I]-5-[3-(p-azidosalicylamide)]allyl-UDPglucose, a photoactive analogue of UDP glucose, which is a substrate for this enzyme. Upon photolysis the 125I-labeled probe was shown to link covalently to the 66 kDa protein. The photoinsertion was competed out by the presence of unlabeled UDPglucose thus suggesting that this protein contains substrate binding site for UDPglucose. Since photoinsertion of the probe to protein of 66 kDa correlates with the molecular mass of the protein visualized upon enzyme purification we postulate that the 66 kDa protein is involved in sterolglucoside synthesis in yeast.

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