Quantitation of proteins solubilized in sodium dodecyl sulfate-mercaptoethanol-tris electrophoresis buffer

Zaman, Zahur; Verwilghen, R. L.

doi:10.1016/0003-2697(79)90110-6

Cited by 103 publications

(32 citation statements)

References 9 publications

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“…Protein concentration was estimated by a Coomassie-blue-G250-binding assay [20] using a commercial standard protein solution (Sigma) containing human albumin and globulin ( 5 : 3). Protein concentration in column effluents was monitored at 280 nm.…”

Section: Polybuffer Removulmentioning

confidence: 99%

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Purification and characterization of trypsin from the poikilotherm Gadus morhua

Ágeirsson

Fox

Bjarnason

1989

European Journal of Biochemistry

176

139

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A serine protease shown to be trypsin was purified from the pyloric caeca of Atlantic cod (Gadus morhua), and resolved into three differently charged species by chromatofocusing (PI 6.6,6.2 and 5.5). All three trypsins had similar molecular mass of 24.2 kDa. N-terminal amino acid sequence analysis of cod trypsin showed considerable similarity with other known trypsins, particularily with dogfish and some mammalian trypsins. The apparent K, values determined at 25 "C for the predominant form of Atlantic cod trypsin towards p-tosyl-L-arginine methyl ester and N-benzoyl-L-arginine p-nitroanilide were 29 pM and 77 pM respectively, which are notably lower values than those determined for bovine trypsin (46 pM and 650 pM respectively). The difference was particularly striking when the amidase activity of the enzymes was compared. Furthermore, the k,,, values determined for the Atlantic cold trypsins were consistently higher than the values determined for bovine trypsin. The higher catalytic efficiency (kJK,,,) of Atlantic cod trypsin as compared to bovine trypsin may reflect an evolutionary adaptation of the poikilothermic species to low environmental temperatures.

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Section: Polybuffer Removulmentioning

confidence: 99%

“…Protein staining was carried out for 30-60 min with 0.5% (massivol.) Coomassie blue G250 treated with 0.5 M perchloric acid [20] and the gels were destained and stored in 7.5% (by vol.) acetic acid.…”

Section: Assays Electrophoresismentioning

confidence: 99%

Purification and characterization of trypsin from the poikilotherm Gadus morhua

Ágeirsson

Fox

Bjarnason

1989

European Journal of Biochemistry

176

139

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“…The percentage breakdown ofeach polypeptide was taken as the loss in 60 min in the area under each peak relative to that present initially. Proteins were estimated after removal of NaDodSO4 (39,40). (41).…”

mentioning

confidence: 99%

Liver mitochondria contain an ATP-dependent, vanadate-sensitive pathway for the degradation of proteins.

Desautels¹,

Goldberg²

1982

Proc. Natl. Acad. Sci. U.S.A.

114

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A large fraction (30-50%) of the various proteins synthesized within isolated rat liver mitochondria were degraded to amino acids within 60 min after synthesis. Incomplete mitochondrial polypeptides resulting from the incorporation of puromycin were degraded even more extensively (80% per hr). Protein breakdown was measured by the appearance of acid-soluble radioactivity and by the disappearance of labeled polypeptides detected on NaDodSO4/polyacrylamide gel electrophoresis. The amino acids generated by proteolysis were transported rapidly out of the mitochondria and no peptide intermediates accumulated in the organelle. This degradative process did not involve lysosomes or lysosomal enzymes and was markedly stimulated by ATP either generated within the mitochondria or supplied exogenously. An inhibitor of respiration (cyanide) or uncouplers of oxidative phosphorylation (oligomycin, dinitrophenol) reduced proteolysis when mitochondria were provided substrates for ATP generation. When exogenous ATP was provided, these agents did not affect proteolysis, but degradation was then sensitive to atractyloside, an inhibitor of adenine nucleotide transport. Vanadate, an inhibitor of various ATPases, blocked proteolysis even in the presence of ATP and caused a marked stabilization of nearly all polypeptide bands. Thus, mitochondria-like bacteria or the cytosol of animal cells-contain a pathway for complete degradation of proteins which seems to selectively remove polypeptides with abnormal structures. Within this organelle, ATP hydrolysis appears necessary for an initial step in this degradative process.The proteins comprising the mitochondria, like other proteins in mammalian cells, are subject to continual turnover (1). It has generally been assumed that mitochondria are degraded within the lysosome (2-5), and under certain conditions whole mitochondria or mitochondrial enzymes have been demonstrated within autophagic vacuoles (2-4). However, different mitochondrial proteins turn over at distinct rates. Outer membrane proteins tend to be degraded at a faster rate than those of the inner membrane (6). Furthermore, individual proteins within the same mitochondrial compartment can also have different turnover rates. In the matrix, several enzymes have tl/2 ranging from 70 min to 1-2 days, whereas the bulk of mitochondrial proteins turn over with a tl/2 of 3-5 days (6-8). Such observations cannot be explained by indiscriminate degradation of this organelle within the lysosome.One possible explanation for this heterogeneity in degradative rates is that proteins may be excreted by the mitochondria for degradation in the cytoplasm or lysosome. Another possibility is that there exists within mitochondria a proteolytic system that selectively hydrolyzes the short-lived enzymes. Several groups have reported proteases associated with the mitochondrial fraction of mammalian cells (9-14). However, an intramitochondrial localization has not been demonstrated definitively for any of these enzymes. The presence in the mitochon...

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“…Plasma membranes (PMs), were prepared by filter lysis and sucrose density centrifugation (Das and Henderson, 1983) from axenically grown Ax2 or gp138-null cells kindly provided by Dr. Urushihara (University of Tsukuba, Japan; Hata et al, 2001). Protein levels were measured using either the Bradford method (Bradford, 1976), after precipitating interfering SDS with 100 mM potassium phosphate, pH 7.2 (Zaman and Verwilghen 1979), or the Lowry method (Lowry et al, 1951) in the presence of 0.1% SDS and bovine serum albumin (Pierce Chemical, Rockford, IL) as a standard.…”

Section: Protein Analysesmentioning

confidence: 99%

Glycoprotein gp130 ofDictyostelium discoideumInfluences Macropinocytosis and Adhesion

Chia

Gomathinayagam

Schmaltz

et al. 2005

MBoC

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Glycoprotein gp130, found on the plasma membrane of Dictyostelium discoideum amoebae, was postulated previously to play a role in phagocytosis. The gene for gp130 was cloned and when translated, yielded a 768 amino acid preproprotein of 85.3 kDa. It had nearly 40% similarity to the 138 kDa family of glycoproteins implicated in sexual cell fusion during macrocyst formation in D. discoideum. The difference between the calculated size and observed M r of 130 kDa on protein gels likely was due to N-glycosylation that was confirmed by lectin blots. Consistent with its surface-exposure, an antibody raised against recombinant protein stained the plasma membrane of D. discoideum amoebae. Gp130 and its transcripts were high during axenic growth of cells, but relatively low during growth on bacteria. The gene for gp130 was disrupted and cell lines lacking the glycoprotein were efficient phagocytes, indicating that gp130 was dispensable for phagocytosis. Gp130-null cells were similar in size to parent DH1 cells, had enhanced macropinocytosis and grew faster to higher densities. They also exhibited weaker cell-substrate adhesion but displayed greater cell-cell cohesion. Collectively, the data indicated that gp130 influenced macropinocytosis and played a role in adhesion during vegetative growth. INTRODUCTIONThe processes of phagocytosis, cell-cell and cell-substrate adhesion share a common initial event of recognition, whether it is of specific ligands or the chemical properties of the partner(s) in the interaction. With the long-term aim of understanding the mechanism of phagocytosis at the molecular level, particularly the early steps of recognition and binding, we have focused on surface-exposed molecules of the phagocytically active amoebae of Dictyostelium discoideum. D. discoideum cells are studied also for the related processes of motility and aggregation. When starved, individual but clonal cells coalesce into multicellular structures that, within a day, differentiate into fruiting bodies called sorocarps. Cell-cell recognition and adhesion are vital aspects of this developmental progression, and researchers have established that cell-surface molecules, including gp24 (Knecht et al., 1987;Loomis, 1988;Brar and Siu, 1993), gp80 (Muller and Gerisch, 1978;Noegel et al., 1986), and gp150 (Gao et al., 1992;Wang et al., 2000), are mediating these interactions (reviewed in Kessin, 2001 andSiu et al., 2004). An understanding of the plasma membrane molecules involved in adhesive events during phagocytosis and motility of vegetative amoebae is emerging with the recent identification of cell-substrate adhesion molecule, sadA (Fey et al., 2002) and the phg1 family of transmembrane 9 proteins (Cornillon et al., 2000;Benghezal et al., 2003).In this study, we present evidence that a plasma membrane glycoprotein gp130 also played a role in cell-substrate adhesion during vegetative growth. Postulated to be a phagocytosis receptor (Chia, 1996), gp130 is possibly the same molecule as gp126, a surface-exposed glycoprotein suggested to ...

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Quantitation of proteins solubilized in sodium dodecyl sulfate-mercaptoethanol-tris electrophoresis buffer

Cited by 103 publications

References 9 publications

Purification and characterization of trypsin from the poikilotherm Gadus morhua

Purification and characterization of trypsin from the poikilotherm Gadus morhua

Liver mitochondria contain an ATP-dependent, vanadate-sensitive pathway for the degradation of proteins.

Glycoprotein gp130 ofDictyostelium discoideumInfluences Macropinocytosis and Adhesion

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