Showing papers on "Pore forming protein published in 1983"

Purification and reconstitution in lipid bilayer membranes of an outer membrane, pore-forming protein of Aeromonas salmonicida.

Abstract: We have purified a major outer membrane protein from Aeromonas salmonicida. This 42-kilodalton protein shared several physical characteristics with enterobacterial porins in that it was noncovalently associated with the peptidoglycan, it was released from the peptidoglycan in the presence of 0.1 M NaCl and sodium dodecyl sulfate, and its mobility on sodium dodecyl sulfate-polyacrylamide gels was dependent on the solubilization temperature before electrophoresis. When added to the aqueous solution bathing a planar bilayer membrane it caused the conductance of the membrane to increase by several orders of magnitude. At lower protein concentrations, single channels with an average conductance of 1.6 nS in 1 M KCl were incorporated into the membrane in a stepwise fashion. Evidence that the protein formed a large, relatively nonselective, water-filled channel was obtained by performing single-channel experiments at different NaCl concentrations and in a variety of different salts. Current through the channel was a linear function of the applied voltage, and no evidence of voltage gating was observed. In addition, we obtained evidence for a 43-kilodalton channel-forming protein in the outer membrane of A. hydrophila with a similar single-channel conductance as the 42-kilodalton protein in 1 M NaCl.

Purification andReconstitution inLipid Bilayer Membranes of anOuterMembrane, Pore-Forming Protein ofAeromonas salmonicida

Abstract: We havepurified amajor outer membrane protein fromAeromonas salmonicida. This42-kilodalton protein shared several physical characteristics with enterobacterial porins inthat itwasnoncovalently associated withthepeptidoglycan,itwasreleased fromthepeptidoglycan inthepresence of0.1M NaCland sodiumdodecyl sulfate, anditsmobility onsodium dodecyl sulfate-polyacrylamide gels wasdependent onthesolubilization temperature before electrophoresis. Whenaddedtotheaqueous solution bathing aplanar bilayer membrane it caused theconductance ofthemembrane toincrease byseveral orders of magnitude. Atlowerprotein concentrations, single channels withanaverage conductance of1.6nSin1M KCIwereincorporated into themembrane ina stepwise fashion. Evidence that theprotein formed alarge, relatively nonselective, water-filled channel wasobtained byperforming single-channel experiments atdifferent NaClconcentrations andinavariety ofdifferent salts. Current through thechannel wasalinear function oftheapplied voltage, andnoevidence of voltage gating wasobserved. Inaddition, weobtained evidence fora43-kilodalton channel-forming protein intheoutermembrane ofA.hydrophila withasimilar single-channel conductance asthe42-kilodalton protein in1M NaCl. Theouter cell envelope ofgram-negative bacteria contains three layers, aninner membrane, apeptidoglycan layer, andanoutermembrane (20). Theouter membrane hasbeenshowntobe abarrier forthepenetration ofawidevariety of compounds (18) while atthesametimepermitting thesize-dependent passage ofsmall molecules (8,16). Thismolecular-sievin g action of