In addition to their well defined role in presentation of processed antigen on the cell surface, class II molecules are able to transduce signals into the cell after binding of ligands. The cytoplasmic regions of class II molecules might function as docking sites for as yet unidentified proteins that are components of this signalling pathway. Here we report on two putative HLA class II associated proteins (PHAPI and PHAPII) which have been purified from the cytosolic fraction of the human lymphoblastoid B-cell line H2LCL using an affinity matrix composed of the synthetic biotinylated cytoplasmic region of the DR2 alpha chain immobilized on avidin agarose. The sequence obtained for PHAPI revealed a novel primary structure with a leucine/isoleucine rich N-terminal region. Protein data and the cDNA sequence obtained for PHAPII agree with the cDNA sequence of SET that has been described recently. Both PHAPI and PHAPII have an extended highly acidic C-terminal region. Based on their primary structure we speculate that PHAPI and PHAPII are involved in the generation of intracellular signalling events that lead to regulation of transcriptional activity after binding of a ligand to HLA class II molecules.

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The cDNA libraries constructed from the human acute lymphoblastic leukemia cell line KM3 in the expression vector lambda gt11, were screened with the anti-CALLA (common acute lymphoblastic leukemia antigen) mAb (monoclonal antibody) J5. The selected J5-positive clone I containing a partial cDNA insert was isolated and sequenced. For completing the cDNA sequence the cDNA libraries were further screened by hybridization with the DIG (digoxigenin)-labelled DNA probe derived from clone I, the 5'-end region was analysed by 5'-RACE (rapid amplification of cDNA ends) using a sequence specific primer. In total a 1639 bp cDNA sequence was determined. The cDNA sequence contains a 1260 bp open reading frame and the untranslated 3'- and 5'-end sides. The 420 residue amino acid sequence, deduced from the cDNA sequence, unexpectedly differs fundamentally from CALLA (CD10) although clones I and II were J5-positive in immuno screening. The mature protein corresponding to the cDNA was isolated and characterized from the KM3 cells using polyclonal antisera raised against the in vitro expressed polypeptide from clone I. The protein is expressed on plasma membrane, in cytosol and is secreted into culture medium, its relative molecular mass was determined to be 55 kDa on SDS-PAGE. The deduced amino acid sequence from cDNA was confirmed by peptide sequences. The new protein contains a basic amino acid rich putative DNA binding domain (b) with a potential nuclear targeting signal, two helix-loop-helix (HLH) motif regions, concurrently EF-hand motifs, an acidic amino acid rich region (a) between the EF-hands, and a leucine zipper (Z) motif. This DNA binding protein therefore is characterized by a linked motif "b/HLH/a/HLH/Z". The protein was designated NEFA: DNA binding/EF-hand/acidic amino acid rich region.

Precise structural studies with Bence-Jones proteins have assumed much greater significance following the suggestion by Edelman and Poulik' that they are polypeptide chains associated with the ey-globulins of patients with multiple myeloma and the confirmation of this suggestion by the findings2 that these myeloma globulins after reduction and alkylation give bands in starch gel electrophoresis which are at the same position as those given by the Bence-Jones protein (which has been similarly reduced and alkylated) from the same patient.This concept has been further strengthened by the findings of Edelman and Gally,3 Putnam,4 Schwartz and Edelman,5 and Mannik and Kunkel.6 It is now accepted that the Bence-Jones proteins correspond in most instances to the light chains of the myelorna protein from the same patient. The light chains are known7 to be important in antibody structure. Structural differences between different Bence-Jones proteins might not only reveal certain features responsible for antibody specificity but might also yield more detailed information concerning the genetic mechanism which determines the synthesis of light chains of antibodies.A study of the amino acid sequence of the Bence-Jones proteins from certain selected patients was therefore undertaken. The patients were selected on the basis of minimal heterogeneity with respect to preliminary chemical studies8 and the physical and immunological studies carried out in the laboratories of Dr. H. G. Kunkel and Dr. G. M. Edelman of this institute.

We report on the production and characterization of eight monoclonal mouse antibodies against the complete human VDAC "Porin 31HL". The antigen used was purified from a total membrane preparation of the transformed human B-lymphocyte cell line H2LCL.In Western blots all eight mAbs react with a single 31-kDa band in solubilized H2LCL membrane preparations thus demonstrating their specificity for the human VDAC 'Torin 31HL". Concerning the epitope specificity we show that all eight mAbs equally react with the N-terminal part of human porin.Moreover, we demonstrate the expression of VDAC in the sarcolemma by indirect immunoenzyme labelling of cryosections of human skeletal muscle applying four of our mAbs. These data support our recent observations on the expression of porin channels in the plasmalemma of different normal and transformed human cell lines.

Hämoglobine bestehen aus zwei Komponenten: dem eisenhaltigen Häm lind Protein (Globin). Das Mol.-Gew. wurde in der Ultrazentrifuge zu 66-68000 ermittelt 1 . Auf dieses Mol.-Gew. bezogen, besteht die Partikel aus vier Fe-Atomen, vier Porphyrinringen und vier Peptidketten, von denen je zwei identisch vorliegen 2 . Die vier Harne sind für alle Ketten identisch. Die Konstitution dieses Anteils wurde von Hans Fischer ermittelt.Im folgenden wird über die vollständige chemische Struktur des Proteins der Hauptkomponente des normalen adulten Humanhämoglobins berichtet. Unsere früheren Arbeiten handelten über die Spaltung des Globins mit Trypsin 3 , Trennung der Spaltprodukte 4 , über eine Partialformel der a-5 und ß-Kette 6 sowie über die Lage der Sequenzlücken 7 .Zur vollständigen Strukturaufklärung mußten wir noch die Sequenzanalyse des sog. a-und /?-Cores sowie des Peptides HT all (62-90 der -Kette) ergänzen:Das -Core (bearbeitet von G. H.) wurde durch Reduktion der SH-bzw. der evtl. vorhandenen S-S-Gruppen nach Swan 8 bzw. Neurath 8 oder durch Reduktion und Carboxymethylierung mit Jodacetamid nach Anfinsen 9 vorbehandelt, der so modifizierte Kettenanteil anschließend chymotryptisch bzw. peptisch hydrolysiert und die über Dowex 1X2 10 isolierten Spaltprodukte der Sequenzanalyse unterworfen.Das ß-CoTe (bearbeitet von N. H.) wurde nach Oxydation der beiden SH-Gruppen mit Perameisensäure 11 mit Pepsin gespalten; die Bruchstücke wurden ebenfalls mittels Dowex 1X2 isoliert.

Porin 31HL was isolated and purified from total membrane preparations of a human B-lymphocyte cell line. The protein showed a single band of apparent molecular mass 31kDa on SDS-PAGE. Reconstitution of the protein into artificial lipid bilayer membranes defines its function as a channel-forming protein. The distribution of single-channel conductances had two maxima of 2.4 and 4.3 nS in IM KC1. The channel formed by Porin 31HL of human B-lymphocytes was found to be voltage-dependent and switched to ion-permeable substates at membrane voltage larger than 20 m V. In the open state the pore exhibited the characteristics of a general diffusion pore because the mobility sequence of the ions inside the pore was similar to that in the bulk aqueous phase. The effective diameter was estimated to be about 1.7nm. The properties of the low conductance state of the channel were studied in detail. In this state the pore favored the passage of cations, in contrast to the open state which favored anions slightly. Monoclonal antibodies against the N-terminal end of Porin 31HL blocked its reconstitution but had otherwise no influence on the channel properties. This result suggested that the amphipathic -helical structure at the N-terminal end is probably not involved in channel gating. The channel-forming properties of Porin 31HL were compared to those of porins isolated from mitochondrial outer membranes and to those of the "maxi chloride channel" observed in the cytoplasmic membrane of several eukaryotic cells.

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