Channel active mammalian porin, purified from crude membrane fractions of human B lymphocytes or bovine skeletal muscle, reversibly binds the stilbene-disulfonate group of the chloride channel blocker DIDS.
Two new aspects of mammalian porin are presented. First, by affinity chromatography we show that channel active human or bovine porin reversibly bind the stilbene-disulfonate group of the chloride channel blocker 4,4'-diisothiocyanatostilbene-2,2'-disulfonate (DIDS). The procedure is suitable for further purification of porin after enrichment by ion exchange chromatography and shows a yield of 24.3%. The data support our recent proposal that VDAC forms part of the ORDIC channel complex which is affected in cystic fibrosis. Second, a purification scheme for mammalian porin is given starting with direct solubilisation of ground bovine skeletal muscle to avoid breaking up tissue. About 130 mg of channel active "Porin 31BM" are enriched from 946 g muscle tissue. Concerning its apparent molecular mass, primary structure, channel activity, channel conductance and voltage dependence the molecule shows high similarity to human porin. "Porin 31BM" is furthermore labelled by antibodies raised against human B lymphocyte derived "Porin 31HL".
Thinnes
F P
FP
Max-Planck-Institut für experimentelle Medizin, Abteilung Immunchemie, Göttingen, Germany.
Flörke
H
H
Winkelbach
H
H
Stadtmüller
U
U
Heiden
M
M
Karabinos
A
A
Hesse
D
D
Kratzin
H D
HD
Fleer
E
E
Hilschmann
N
N
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000378:metabolism* / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D001402:B-Lymphocytes / Q000737:chemistry*; D015153:Blotting, Western; D002417:Cattle; D018118:Chloride Channels / Q000737:chemistry / Q000187:drug effects / Q000302:isolation & purification / Q000378:metabolism*; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008051:Lipid Bilayers; D008565:Membrane Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009132:Muscles / Q000737:chemistry*; D018272:Porins; D050995:Voltage-Dependent Anion Channel 1; D050994:Voltage-Dependent Anion Channels
D018118:Chloride Channels; D008051:Lipid Bilayers; D008565:Membrane Proteins; D018272:Porins; C494762:VDAC1 protein, human; D050994:Voltage-Dependent Anion Channels; D050995:Voltage-Dependent Anion Channel 1; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid