PubMed · 4230100
The source did not provide an abstract. Follow the original record for more information.
Explore connections, maps & timelines
W R Swaim. 1968. Plasminogen assay.. https://pubmed.ncbi.nlm.nih.gov/4230100/
Cite the original work for its findings. Save a collection to share your selection of sources.
KEEP EXPLORING
The actin-ADP-ribosylating binary Clostridium botulinum C2 toxin consists of two individual proteins, the binding/translocation component C2II and the enzyme component C2I. To elicit its cytotoxic action, C2II binds to a receptor on the cell surface and mediates cell entry of C2I via receptor-mediated endocytosis. Here we report that binding of C2II to the surface of target cells requires cleavage of C2II by trypsin. Trypsin cleavage causes oligomerization of the activated C2II (C2IIa) to give SDS-stable heptameric structures, which exhibit a characteristic annular or horseshoe shape and form channels in lipid bilayer membranes. Cytosolic delivery of the enzyme component C2I is blocked by bafilomycin but not by brefeldin A or nocodazole, indicating uptake from an endosomal compartment and requirement of endosomal acidification for cell entry. In the presence of C2IIa and C2I, short term acidification of the extracellular medium (pH 5.4) allows C2I to enter the cytosol directly. Our data indicate that entry of C2 toxin into cells involves (i) activation of C2II by trypsin-cleavage, (ii) oligomerization of cleaved C2IIa to heptamers, (iii) binding of the C2IIa oligomers to the carbohydrate receptor on the cell surface and assembly with C2I, (iv) receptor-mediated endocytosis of both C2 components into endosomes, and finally (v) translocation and release of C2I into the cytosol after acidification of the endosomal compartment.
In higher vertebrates, reactive gliosis resulting from injury to the central nervous system (CNS) is characterized by a rapid increase in immunoreactivity (IR) to glial fibrillary acidic protein (GFAP). Little is known about the extracellular signals that initiate the increase in GFAP-IR following CNS injury. We demonstrated recently [T.H. Oh, G.J. Markelonis, J.R. Von Visger, B. Baik, M.T. Shipley, Acidic pH rapidly increases immunoreactivity of glial fibrillary acidic protein in cultured astrocytes, Glia 13 (1995) 319-322] that a rapid increase in GFAP-IR can be evoked in mature astrocyte cultures by exposing the cells to an acidic medium. We investigated the intracellular pathway(s) involved in initiating increased GFAP-IR, a hallmark of reactive astrocytes. The increase in GFAP-IR produced by exposure to acidic medium was blocked by pretreatment with nickel ions, by such blockers of L-type calcium channels as nifedipine, verapamil and diltiazem, by calpain inhibitor I, or by the intracellular calcium chelator, BAPTA-AM. At physiological pH, treatment with the calcium ionophore, A23187, resulted in increased GFAP-IR which could be blocked by pretreatment with calpain inhibitor I. Astrocytes exposed to low pH exhibited a marked increase in a GFAP fragment with a molecular weight of 48 kDa. In astrocytes exposed to acidic medium, alpha-fodrin, a selective endogenous substrate of calpain, was also found to be hydrolyzed producing fragments with molecular weights of 120-150 kDa. As anticipated, pretreatment with calpain inhibitor I prevented the proteolytic degradation of both GFAP and alpha-fodrin in these samples. These results suggest that the initial increase in GFAP-IR after CNS injury appears to be linked to Ca(++) influx, and is mediated further by a proteolytic process that seemingly involves the activation of the calcium-dependent protease, calpain I.
The folding kinetics of the multidomain ribosomal protein L9 were studied using pH jump stopped-flow fluorescence and circular dichroism (CD) in conjunction with guanidine hydrochloride (GdnHCl) jump stopped-flow CD experiments. Equilibrium CD and 1D (1)H NMR measurements demonstrated that the C-terminal domain unfolds below pH 4 while the N-terminal domain remains fully folded. Thus, the N-terminal domain remains folded during the pH jump experiments. The folding rate constant of the C-terminal domain was determined to be 3.5 s(-1) by pH jump experiments conducted in the absence of denaturant using stopped-flow CD and fluorescence. CD-detected GdnHCl jump measurements showed that the N- and C-terminal domains fold independently each by an apparent two-state mechanism. The folding rate constant for the N-terminal domain and the C-terminal domain in the absence of denaturant were calculated to be 760 and 4. 7 s(-1), respectively. The good agreement between the pH jump and the denaturant concentration jump experiments shows that the folding rate of the C-terminal domain is the same whether or not the N-terminal domain is folded. This result suggests that the slow folding of the C-terminal domain is not a consequence of unfavorable interactions with the rest of the protein chain during refolding. This is an interesting result since contact order analysis predicts that the folding rate of the C-terminal domain should be noticeably faster. The folding rate of the isolated N-terminal domain was also measured by stopped-flow CD and was found to be the same as the rate for the domain in the intact protein.