Channel active mammalian porin, purified from crude membrane fractions of human B lymphocytes and bovine skeletal muscle, reversibly binds adenosine triphosphate (ATP).
A new aspect of mammalian porin (mammalian VDAC = mammalian voltage-dependent anion channel) is presented: channel active VDAC binds adenosine triphosphate (ATP) in the absence of Ca2+. Channel active "Porin 31HL" or "Porin 31BM", enriched from crude membranes of human B lymphocytes or whole cell lysates of bovine skeletal muscle, respectively, was bound to a nine atoms spacer ATP-agarose at pH 7.4 or 5.0 and reeluted from the resin by 10 mM ATP disodium salt. Furthermore, channel active "Porin 31BM" was labelled by [32P]ATP in a 1:1 stoichiometric relation. Binding of ATP to human porin was confirmed by studying the interaction of the synthetic porin fragment Type-1/Ac-35, comprising the putative nucleotide binding site G Y G F G, with trinitrophenyl-ATP (TNT-ATP) by scanning fluorometry. Peptide/TNP-ATP complexes clearly show enhancement of fluorescence intensity and a spectral shift of the fluorescence maximum. In a control experiment, using a porin fragment lacking the putative nucleotide binding site, no change of fluorescence emission was observed. Further confirmation for ATP binding by human VDAC arose from an autoradiographic experimental approach: the porin fragment Type-1/Ac-35 could be labelled by [32P]ATP, while a second porin fragment ending immediately before the putative nucleotide binding site could not; nor could a synthetic non porin peptide.
Flörke
H
H
Max-Planck-Institut für experimentelle Medizin, Abteilung Immunchemie, Göttingen, Germany.
Thinnes
F P
FP
Winkelbach
H
H
Stadtmüller
U
U
Paetzold
G
G
Morys-Wortmann
C
C
Hesse
D
D
Sternbach
H
H
Zimmermann
B
B
Kaufmann-Kolle
P
P
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001402:B-Lymphocytes / Q000166:cytology / Q000378:metabolism*; D015153:Blotting, Western; D002417:Cattle; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D005470:Fluorometry; D006801:Humans; D007473:Ion Channels; D008565:Membrane Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D008928:Mitochondria; D008969:Molecular Sequence Data; D008970:Molecular Weight; D018482:Muscle, Skeletal / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D010446:Peptide Fragments; D018272:Porins / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D013050:Spectrometry, Fluorescence; D050995:Voltage-Dependent Anion Channel 1; D050994:Voltage-Dependent Anion Channels
C009587:2',3'-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate; D007473:Ion Channels; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D010446:Peptide Fragments; D018272:Porins; C494762:VDAC1 protein, human; D050994:Voltage-Dependent Anion Channels; D000255:Adenosine Triphosphate; D050995:Voltage-Dependent Anion Channel 1