A proteinase from human erythrocyte membranes

Moore, Gerald L.; Kocholaty, Walter F.; Cooper, Donald A.; Gray, John; Robinson, Stephen L.

doi:10.1016/0005-2744(70)90185-3

Cited by 73 publications

(16 citation statements)

References 10 publications

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“…Protein degradation in human erythrocyte membrane has also been reported, mostly for ghosts incubated for many hours (21)(22)(23)(24)(25). Various Ca2e-dependent and Ca2e-independent proteases have been identified and isolated from human erythrocytes (26)(27)(28)(29). Degradation of transmembrane proteins, band 3 and glycophorin, as shown by immunochemical methods, occurs in the Ca2+-loaded, intact human erythrocyte in addition to formation of high Mr proteins in these cells (30).…”

Section: Discussionmentioning

confidence: 99%

Membrane-mobility agent-promoted fusion of erythrocytes: fusibility is correlated with attack by calcium-activated cytoplasmic proteases on membrane proteins.

Kosower¹,

Glaser²,

Kosower³

1983

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

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Rat, but not human, erythrocytes undergo fusion promoted by the membrane-mobility agent 2-(2-methoxyethoxy)-ethyl cis-8-(2-octylcyclopropyl)octanoate (A2C). The difference in behavior is correlated with rat erythrocyte membrane protein degradation caused by Ca2+-activated proteases. The human erythrocyte is deficient in such protease activity. Membrane protein degradation is a necessary, but not sufficient, requirement for membrane fusion. Membrane protein degradation probably releases membrane components from certain constraints. In addition, the motion of membrane components precedes fusion and must be promoted by reagents such as A2C, leading to the creation of fusion-potent lipid areas. This sequence of chemical and physical events occurs in other fusion processes.Membrane fusion is a ubiquitous cellular process, mediating such phenomena as fertilization, exocytosis, endocytosis, phagocytosis, and turnover of intracellular organelles (1, 2). Fusion can be induced experimentally by certain viruses (3) and chemical agents (4, 5). Membrane-mobility agents are long-chain esters designed to promote motion of molecules in cell membranes. Membrane-mobility agents, which enter cells via the small particles formed on dispersion in aqueous medium, promote the lateral mobility of certain ligand-membrane receptor complexes and inhibit cytokinesis in some types of cells (5-9). Membrane-mobility agents, especially 2-(2-methoxyethoxy)ethyl cis-8-(2-octylcyclopropyl)octanoate (A2C), are efficient promoters of cell fusion; the results obtained using a defined agent such as A2C have allowed the identification of stages in the overall fusion process and the formulation of a molecular mechanism for membrane fusion (10-13).We have previously found differences in the occurrence of A2C-induced fusion among various mammalian non-nucleated erythrocytes as well as among avian nucleated erythrocytes (12, 13): erythrocytes of some species fuse easily, whereas those of others do not. "Mixed fusion," that between fusible erythrocytes from different species, is promoted by A2C. Moreover, the potential for fusion is a transferable characteristic, fusion being induced by A2C in otherwise nonfusible cells if mixed with fusible erythrocytes (13). The molecular basis for the differences observed among the erythrocytes of various species and for the transfer of fusibility has not yet been clarified.We now show that the difference between fusible rat erythrocytes and the nonfusible human cells is correlated with proteolytic activity; degradation of rat cell membrane proteins is caused by a Ca2'-activated cytoplasmic protease(s). The human erythrocyte is deficient in this protease activity. Membrane protein degradation is shown to be a necessary, but not sufficient, prerequisite for membrane fusion. Release of membrane components from constraints imposed by the cytoskeleton, allowing membrane component mobility, is proposed as one of the steps in the fusion process. Furthermore, the mobility of the membrane components must be promoted by...

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Section: Discussionmentioning

confidence: 99%

Membrane-mobility agent-promoted fusion of erythrocytes: fusibility is correlated with attack by calcium-activated cytoplasmic proteases on membrane proteins.

Kosower¹,

Glaser²,

Kosower³

1983

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

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“…IL, respectively, by proteolysis during the preparation of the sialoglycoproteins . Several neutral (3,15,26,27,41,42) and acid proteases (14,17,19,28,31) have been detected in preparations of erythrocyte ghosts, arising probably from contaminating leukocytes (15). Some of the neutral proteases have essential sulfhydryl groups (27) and perhaps are partially inactivated by STT like other enzymes with essential sulfhydryls (21,29,30).…”

Section: Discussionmentioning

confidence: 99%

Isolation and partial characterization of the sialoglycoprotein fraction of murine erythrocyte ghosts.

Sarris¹,

Palade²

1982

The Journal of Cell Biology

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With the lithium diiodosalicylate (LIS) extraction-phenol partition method, we have isolated a sialoglycoprotein fraction from DBA/2 mouse erythrocyte ghosts . We have demonstrated that the Laemmeli system for SDS PAGE can resolve this fraction into four monomers of which two (gp-2.1 and gp-3 .1) appear to be authentic, whereas the other two (gp-2 .2 and gp-3 .2) are probably generated from gp-2 .1 and gp-3 .1, by limited proteolysis during the isolation procedure. All four components contain O-acetylated neuraminic acid residues, can be stained with Periodic acid-Schiff reagent (PAS) and with Coomassie Brilliant Blue (CB), and can be radioiodinated with the lactoperoxidase-glucose oxidase (LPO-GO) method . All monomers but especially gp-2 .1 and gp-3 .1 generate characteristic aggregates during solubilization in SDS. The aggregation is enhanced by boiling at high concentrations, and can be reversed by boiling at low concentrations . In addition, the fraction contains a diffuse component present also in ghosts which stains poorly with CB and with PAS and cannot be radioiodinated by the LPO-GO technique. SDS PAGE in the Steck and Yu gel system does not give an accurate separation of the sialoglycoprotein monomers .In our previous work on murine erythrocyte ghosts, we detected three sialoglycoproteins by Periodic acid-Schiff reagent (PAS) staining after SDS PAGE carried out according to Steck and Yu (37), and we presented evidence for the existence and preferential localization of O-acetylated sialyl residues on two of these three sialoglycoproteins (32) . When the sialoglycoproteins were subsequently analyzed on Laemmli gels (20), however, we obtained a very different pattern. The discrepancy raised the following questions : Which of the visible bands are sialoglycoprotein monomers and which are aggregates? Which of the gel systems truly separates the monomers? And, if polymerization occurs, what are the conditions that can prevent it in vitro?Here we partially answer these questions. We eluted all resolved components from each of the two gel systems and reran each component in the same or the alternative system . Each band will be named by a prefix gp, (for glycoprotein), and a suffix: S to signify initial separation on a Steck and Yu gel ; or L to signify separation on a Laemmli gel. The prefixes will always be used in the text but may be omitted from the figures . Gel Electrophoresis SDS PAGE was performed by solubilizing the proteins in 62 .5 mM Tris-HCI, pH 6.8, 3% wt/vol SDS, 2 M urea, 2 mM EDTA, and either 20 mM dithiothreitol (DTT) or 1 .5% (vol/vol) ß-mercaptoethanol, heating the mixture in a boiling water bath for 5 min, and then separating its components according to Laemmli (20) . Some analyses were also performed on Steck and Yu gels (37) as in Sarris and Palade (32) by solubilizing the preparations in 10 mM Tris-HCI, pH 8.0, 1 mM EDTA, 3% (wt/vol) SDS, 1.5% (vol/vol) ß-mercaptoethanol and 6% (vol/ vol) glycerol, and heating them in a boiling water bath for 5 min before electrophoretic se...

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“…The protein contents of the samples were determined by the method of Lowry et al (32) after precipitation with trichloroacetic acid (TCA) because Tris buffer interfered with the colorimetric reaction. ENZYME ASSAYS: Proteolytic activity was assayed according to the method of Moore et al (35) using Azocoll as a substrate. This sensitive assay is useful in screening for proteolytic activity, since Azocoll is a general substrate which can be hydrolyzed by all known proteolytic enzymes.…”

Section: Methodsmentioning

confidence: 99%

Purification from black widow spider venom of a protein factor causing the depletion of synaptic vesicles at neuromuscular junctions.

Frontali¹,

Ceccarelli²,

Gorio³

et al. 1976

The Journal of Cell Biology

220

139

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The aqueous extract of the venom glands of black widow spiders was fractionated on a column of Sephadex G-200 and then on a column of DEAE-Sephadex A-50, pH 8.2. A protein fraction was obtained that caused a great increase in the frequency of occurrence of miniature end plate potentials at the frog neuromuscular junction, and caused swelling of the nerve terminals and depleted them of their vesicles. The fraction consists of at least four protein components that are similar in their molecular weights (about 130,000) and isoclectric points (ranging from pH 5.2 to 5.5) and are immunologicaUy indistinguishable. It contains no sugar residues and has little or no lipolytic or proteolytic activity. The fraction is toxic to mice and is different from the fractions that act on houseflies, the crayfish stretch receptor and the cockroach heart. It seems pure enough to warrant a detailed study of its site and mode of action.The physiological effects of the extract of the venom glands of the black widow spider, Latrodectus mactans, particularly the variety tredecimguttatus, have been studied extensively and the primary effects seem to be exerted on the nervous system (2-5, 7, 8, 12, 13, 15-18, 20-23, 30, 31, 33, 36-40, 43). Some of the active agents are proteins (18), and they seem to act in at least two basic ways: (a) to depolarize the cell bodies of some neurons and (b) to cause the release of neurotransmitters from a variety of nerve endings. For example, the extract depolarizes the cell body of the crayfish stretch receptor (S. Obara and A. Mauro, unpublished data) and induces a discharge of impulses in the axon (26). This depolarizing action of the extract may be responsible for its ability to induce a discharge of action potentials in the abdominal ganglion of the cockroach (5, 17).In addition to depolarizing excitable cells, the extract causes a release of transmitter from cholinergic nerve endings in brain (21, 24), sympathetic ganglia (38, 39), and Torpedo electric tissue (25), and from adrenergic nerve endings in the iris and other tissues (20,22,24). At neuromuscular junctions, the extract increases the frequency of occurrence of miniature end plate potentials, and blocks neuromuscular transmission. The neuromuscular effects have been observed at the cholinergic junctions of frogs and mammals (31, 37), at adrenergic nerve endings (vas deferens) in mammals (27), at both the inhibitory (3,-aminobutyrate) 462

show abstract

A proteinase from human erythrocyte membranes

Cited by 73 publications

References 10 publications

Membrane-mobility agent-promoted fusion of erythrocytes: fusibility is correlated with attack by calcium-activated cytoplasmic proteases on membrane proteins.

Membrane-mobility agent-promoted fusion of erythrocytes: fusibility is correlated with attack by calcium-activated cytoplasmic proteases on membrane proteins.

Isolation and partial characterization of the sialoglycoprotein fraction of murine erythrocyte ghosts.

Purification from black widow spider venom of a protein factor causing the depletion of synaptic vesicles at neuromuscular junctions.

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