Friedrich P. Thinnes

LENTIL LECTIN ENRICHED MICROSOMES FROM THE PLASMA MEMBRANE OF THE HUMAN B-LYMPHOCYTE CELL LINE H2LCL CARRY A HEAVY LOAD OF TYPE-1 PORIN

Using an established biochemical approach, five subcellular fractions of human B lymphocytes were prepared by differential centrifugation. Crude membranes were passed over a lentil lectin column to enrich carbohydrate-coated cell surface microsomes. The lectin-bound fraction contained a high amount of plasma membrane-derived microsomes as indicated by cell surface markers. All subcellular fractions in Western blots proved to contain distinct but variable amounts of porin. There was a strong increase in porin content from crude membranes to plasma membrane-derived vesicles. The porin content of this fraction appeared to be higher than that of mitochondria. In the final step the plasma membrane-derived microsome fraction proved to be devoid of contamination by outer mitochondrial membranes, as revealed by antibodies against the established markers MAO B and Tom20 applied in Western blots. These data prove the extramitochondrial expression of human type-1 porin/ type-1 VDAC.

Mitochondria-derived and extra-mitochondrial human type-1 porin are identical as revealed by amino acid sequencing and electrophysiological characterisation.

Abstract In mammalian cells porin channels are localised in both mitochondrial outer membranes and extra-mitochondrial membranes. We isolated mitochondria-derived porin of a human lymphoblastoid B cell line, determined its amino acid sequence and characterised its channel properties. Interestingly, the amino acid sequence of this porin preparation and, correspondingly, its electrophysiological characteristics in a reconstituted system were identical to those of ‘Porin 31HL’, the human type-1 porin purified from a crude membrane preparation of the same cell line using a different purification protocol. The results raise questions about targeting, insertion and orientation of human type-1 porin in different membranes.

Ist das HLA-DR-assoziierte Glycoprotein p31 ein ubiquitäres Molekül?

: The glycoprotein p31 (also called Ii, In, M1, Dr gamma, XM 1) has been shown up to now only in the membranes of B lymphocytes as well as of epidermal and endothelial cells, where it is always accompanied by antigens of the HLA-DR type. It is therefore called HLA-DR-associated protein. As we show here, the membranes of muscle, liver and brain contain protein molecules with the relative molecular mass Mr = 31 000-33 000 and an isoelectric point around 7.5. These parameters correspond to those of the p31 of B lymphocytes. These molecules, as well as the p31 of B lymphocytes, can be concentrated by ion exchange chromatography. In two-dimensional electropherograms they are identical to those of B lymphocytes. It can therefore be assumed that the po 31 is not really associated with the HLA-DR antigens but is a ubiquitous molecule.