[40] Isolation of goblet cell apical membrane from tobacco hornworm midgut and purification of its vacuolar-type ATPase

Wieczorek, Helmut; Cioffi, Moira; Klein, Uwe; Harvey, William R.; Schweikl, Helmut; Wolfersberger, Michael G.

doi:10.1016/0076-6879(90)92098-x

Cited by 72 publications

(57 citation statements)

References 10 publications

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“…Immunoprecipitation was performed with the protein G Immunoprecipitation kit (Sigma) following the instructions of the manufacturer. Other methods Protein concentrations were determined by the Amido Black method (Wieczorek et al, 1990), and SDS polyacrylamide gel electrophoresis was performed according to Laemmli (Laemmli, 1970). Determination of chitin synthase activity was performed as described by Zimoch et al .…”

Section: Immunological Methodsmentioning

confidence: 99%

A chymotrypsin-like serine protease interacts with the chitin synthase from the midgut of the tobacco hornworm

Broehan

Zimoch

Wessels

et al. 2007

Journal of Experimental Biology

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The chitin portion of the peritrophic matrix in the midgut of the tobacco hornworm, Manduca sexta, is produced by chitin synthase 2 (CHS2), a transmembrane family II glycosyltransferase, located at the apical tips of brush border microvilli. To look for proteins that potentially interact with CHS2, we performed yeast twohybrid screening, identifying a novel chymotrypsin-like protease (CTLP1) that binds to the extracellular carboxyterminal domain of CHS2. The occurrence of this interaction in vivo is supported by co-localization and coimmunoprecipitation data. Based on our findings we propose that chitin synthesis is controlled by an intestinal proteolytic signalling cascade linking chitin synthase activity to the nutritional state of the larvae.

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Section: Immunological Methodsmentioning

confidence: 99%

A chymotrypsin-like serine protease interacts with the chitin synthase from the midgut of the tobacco hornworm

Broehan

Zimoch

Wessels

et al. 2007

Journal of Experimental Biology

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“…N-terminal Protein Sequencing-V-ATPase was isolated from the goblet cell apical membranes of larval M. sexta midgut as described previously (8,9). Three hundred g of purified V-ATPase were subjected to preparative SDS-PAGE and stained with Coomassie Blue.…”

Section: Methodsmentioning

confidence: 99%

“…Isolation of V-ATPase from partially and from highly purified goblet cell apical membranes of the M. sexta larval midgut as well as from Malpighian tubules, protein determination by Amido Black, SDS-PAGE, [ 14 C]DCCD labeling, Western blotting, and immunostaining were performed as described previously (8,9,23,24). V-ATPase was assayed as enzyme activity sensitive to 1 M bafilomycin B1 (Fluka) according to Wieczorek et al (9).…”

Section: Methodsmentioning

confidence: 99%

A Novel Insect V-ATPase Subunit M9.7 Is Glycosylated Extensively

Merzendorfer

Huss

Schmid

et al. 1999

Journal of Biological Chemistry

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Plasma membrane V-ATPase isolated from midgut and Malpighian tubules of the tobacco hornworm, Manduca sexta, contains a novel prominent 20-kDa polypeptide. Based on N-terminal protein sequencing, we cloned a corresponding cDNA. The deduced hydrophobic protein consisted of 88 amino acids with a molecular mass of only 9.7 kDa. Immunoblots of the recombinant 9.7-kDa polypeptide, using a monoclonal antibody to the 20-kDa polypeptide, confirmed that the correct cDNA had been cloned. The 20-kDa polypeptide is glycosylated, as deduced from lectin staining. Treatment with N-glycosidase A resulted in the appearance of two additional protein bands of 16 and 10 kDa which both were immunoreactive to the 20-kDa polypeptidespecific monoclonal antibody. Thus, extensive N-glycosylation of the novel V o subunit M9.7 accounts for half of its molecular mass observed in SDS-polyacrylamide gel electrophoresis. M9.7 exhibits some similarities to the yeast protein Vma21p which resides in the endoplasmic reticulum and is required for the assembly of the V o complex. However, as deduced from immunoblots as well as from activities of the V-ATPase and endoplasmic reticulum marker enzymes in different membrane preparations, M9.7 is, in contrast to the yeast polypeptide, a constitutive subunit of the mature plasma membrane V-ATPase of M. sexta. Hϩ V-ATPases are a class of ion transport proteins that couple ATP hydrolysis to the movement of protons across membranes. In endomembranes they function, in concert with chloride channels, as acidifiers of intracellular compartments, whereas in plasma membranes their roles are dependent on the cell type. V-ATPases consist of a peripheral V 1 complex, which is responsible for the hydrolysis of ATP, and a membranebound V o complex which is responsible for the translocation of protons. Although the subunit composition may depend upon the source of the enzyme, at least seven subunits of the V 1 complex, subunits A to G, appear to be universal V-ATPase components (1). By contrast, the subunit composition of the V o complex is less clear. There is no doubt that a 16 -17-kDa proteolipid, the proton "channel," is a major constituent of the V o complex. A membrane-associated subunit in the 40-kDa range and an ϳ100-kDa transmembrane subunit may be two additional essential V o components (1). Recently, a novel 9.2-kDa membrane sector-associated polypeptide was reported from bovine chromaffin granules (2). Its sequence and structure show some similarity to Vma21p, a yeast protein involved in the assembly of the V-ATPase; whether or not it is a constitutive V-ATPase subunit remains an open question.In the larval midgut epithelium of the model insect, Manduca sexta (Lepidoptera, Sphingidae), a plasma membrane V-ATPase is present in the apical membranes of goblet cells where it energizes the alkalinization of the gut lumen to a pH of more than 11 (3). For the V 1 complex, amino acid sequences of five insect subunits A, B, E, F, and G have been deduced from cloned cDNAs (4), and evidence for the existence of subuni...

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“…As a control, the inhibition of the V-ATPase activity by the established inhibitor archazolid A was tested in parallel assays. 17 Inorganic phosphate produced in the assays of V-ATPase was measured according to the protocol of Wieczorek et al 35 ( Table 2).…”

mentioning

confidence: 99%

Archazolid A-15-O-β-d-glucopyranoside and iso-Archazolid B: Potent V-ATPase Inhibitory Polyketides from the Myxobacteria Cystobacter violaceus and Archangium gephyra

et al. 2011

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[40] Isolation of goblet cell apical membrane from tobacco hornworm midgut and purification of its vacuolar-type ATPase

Cited by 72 publications

References 10 publications

A chymotrypsin-like serine protease interacts with the chitin synthase from the midgut of the tobacco hornworm

A chymotrypsin-like serine protease interacts with the chitin synthase from the midgut of the tobacco hornworm

A Novel Insect V-ATPase Subunit M9.7 Is Glycosylated Extensively

Archazolid A-15-O-β-d-glucopyranoside and iso-Archazolid B: Potent V-ATPase Inhibitory Polyketides from the Myxobacteria Cystobacter violaceus and Archangium gephyra

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