Isolation of separate apical, lateral and basal plasma membrane from cells of an insect epithelium. A procedure based on tissue organization and ultrastructure

Cioffi, Moira; Wolfersberger, Michael G.

doi:10.1016/0040-8166(83)90050-2

Cited by 68 publications

(31 citation statements)

References 12 publications

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“…The preparation of highly purified goblet cell apical membranes followed published protocols (13,20). SDS-PAGE, Western blotting on nitrocellulose membranes, and immunostaining were performed as described previously (13,21).…”

Section: Methodsmentioning

confidence: 99%

Concanamycin A, the Specific Inhibitor of V-ATPases, Binds to the Vo Subunit c

Huss

Ingenhorst

König

et al. 2002

Journal of Biological Chemistry

254

244

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Section: Methodsmentioning

confidence: 99%

Concanamycin A, the Specific Inhibitor of V-ATPases, Binds to the Vo Subunit c

Huss

Ingenhorst

König

et al. 2002

Journal of Biological Chemistry

254

244

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“…Cioffi and Harvey (Cioffi and Harvey, 1981) showed that the portasome-containing goblet cell apical membranes in posterior midgut do not enclose mitochondria (which would contaminate prospective isolates); nevertheless, posterior midgut transports K + . Based on this information the K + -pump-containing goblet cell apical membrane (GCAM) was isolated by a novel assay based on ultrastructural features, mainly portasomes (Cioffi and Wolfersberger, 1983;Harvey et al, 1983). Dow et al confirmed that the K + -pump is on the GCAM by X-ray microanalysis (see Fig.…”

Section: K + Pumps In Insect Ion-transporting Epitheliamentioning

confidence: 99%

“…5). Although Cioffi, Wolfersberger and Harvey had isolated pure GCAM vesicles and knew that they contained the long sought K + pump (Cioffi and Wolfersberger, 1983;Harvey et al, 1983), they were frustrated because two days work yielded sufficient enzyme for only two or three activity assays. The impasse was broken when Helmut Wieczorek appeared at their door; he had been trying to isolate the K + -ATPase from blowfly labella and had developed a micro assay that enabled one to do hundreds of assays on a tiny sample.…”

Section: K + Pumps In Insect Ion-transporting Epitheliamentioning

confidence: 99%

“…AeNHE3 (Brett's NHE2) from Aedes aegypti was the first insect NHE to be cloned and its location identified in mosquitoes and characterized in yeast and fibroblasts (Pullikuth et al, 2006 Cioffi and Wolfersberger (Cioffi and Wolfersberger, 1983)]. pm, peritrophic membrane; CCAM, columnar cell apical membrane; LM, lateral membrane; GCAM, goblet cell apical membrane; BM, basal membrane; MV, microvilli; M, mitochondrion; SJ, septate junction; GC, goblet cavity; AMP, apical membrane projection; P, portasome (equivalent to V 1 sector of H + V-ATPase); BI, basal infolding; BL, basal lamina.…”

Section: Genomes To the Rescuementioning

confidence: 99%

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Voltage coupling of primary H+ V-ATPases to secondary Na+- or K+-dependent transporters

Harvey

2009

Journal of Experimental Biology

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SUMMARY This review provides alternatives to two well established theories regarding membrane energization by H+ V-ATPases. Firstly, we offer an alternative to the notion that the H+ V-ATPase establishes a protonmotive force (pmf) across the membrane into which it is inserted. The term pmf, which was introduced by Peter Mitchell in 1961 in his chemiosmotic hypothesis for the synthesis of ATP by H+ F-ATP synthases, has two parts, the electrical potential difference across the phosphorylating membrane, Δψ, and the pH difference between the bulk solutions on either side of the membrane, ΔpH. The ΔpH term implies three phases – a bulk fluid phase on the H+ input side, the membrane phase and a bulk fluid phase on the H+ output side. The Mitchell theory was applied to H+ V-ATPases largely by analogy with H+ F-ATP synthases operating in reverse as H+ F-ATPases. We suggest an alternative, voltage coupling model. Our model for V-ATPases is based on Douglas B. Kell's 1979 `electrodic view' of ATP synthases in which two phases are added to the Mitchell model – an unstirred layer on the input side and another one on the output side of the membrane. In addition, we replace the notion that H+ V-ATPases normally acidify the output bulk solution with the hypothesis, which we introduced in 1992, that the primary action of a H+ V-ATPase is to charge the membrane capacitance and impose a Δψ across the membrane; the translocated hydrogen ions (H+s) are retained at the outer fluid–membrane interface by electrostatic attraction to the anions that were left behind. All subsequent events, including establishing pH differences in the outside bulk solution, are secondary. Using the surface of an electrode as a model, Kell's`electrodic view' has five phases – the outer bulk fluid phase, an outer fluid–membrane interface, the membrane phase, an inner fluid–membrane interface and the inner bulk fluid phase. Light flash,H+ releasing and binding experiments and other evidence provide convincing support for Kell's electrodic view yet Mitchell's chemiosmotic theory is the one that is accepted by most bioenergetics experts today. First we discuss the interaction between H+ V-ATPase and the K+/2H+ antiporter that forms the caterpillar K+ pump, and use the Kell electrodic view to explain how the H+s at the outer fluid–membrane interface can drive two H+ from lumen to cell and one K+ from cell to lumen via the antiporter even though the pH in the bulk fluid of the lumen is highly alkaline. Exchange of outer bulk fluid K+ (or Na+) with outer interface H+ in conjunction with (K+ or Na+)/2H+ antiport, transforms the hydrogen ion electrochemical potential difference, \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\overline{{\mu}}_{\mathrm{H}}\) \end{document}, to a K+electrochemical potential difference, \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\overline{{\mu}}_{\mathrm{K}}\) \end{document} or a Na+electrochemical potential difference, \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\overline{{\mu}}_{\mathrm{Na}}\) \end{document}. The \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\overline{{\mu}}_{\mathrm{K}}\) \end{document} or \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\overline{{\mu}}_{\mathrm{Na}}\) \end{document} drives K+- or Na+-coupled nutrient amino acid transporters (NATs), such as KAAT1(K+ amino acid transporter 1), which moves Na+ and an amino acid into the cell with no H+s involved. Examples in which the voltage coupling model is used to interpret ion and amino acid transport in caterpillar and larval mosquito midgut are discussed.

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“…Binding of XnGroEL-BBMV were prepared from dissected gut of fourth and fifth instar larvae by MgCl 2 precipitation, as described previously (20). For binding assay, 20 g of BBMV protein was incubated with 10 g of XnGroEL or variant proteins in a total volume of 30 l and incubated at 4°C for 30 min followed by centrifugation at 12,000 ϫ g for 5 min in cold to remove the unbound protein.…”

Section: Preparation Of Brush Border Membrane Vesicles (Bbmv) Andmentioning

confidence: 99%