1. RNA polymerase from Escherichia coli is selectively and strongly retained by a heparin-substituted agarose and can be eluted therefrom by a neutral buffer containing 0.6 M salt. The method is applicable to relatively crude preparations of the enzyme on a preparative scale giving highly purified RNA polymerase in excellent yield. The enzyme obtained by this procedure shows the highest specific activity so far reported and is pure and enriched in factor CJ as indicated by dodecylsulfate gel electrophoresis.2. Based on the differential affinity of the subunits of the enzyme for the heparin-carrying gel matrix, a method for separation of CI, p' + p and 0 subunits by application of urea and salt-containing buffers is described. Upon recombination and dialysis with urea-free buffer 40-50 % of the enzyme activity is restored.The linear acid mucopolysaccharide heparin is able to interact strongly with several proteins by virtue of its special surface charge distribution. Probably by competition with nucleic acids [l], the polymer inhibits ribonucleases [2 -41 and bacterial and eukaryotic DNA-dependent RNA polymerases, the latter by interfering with the initiation of transcription [4-61. In one case, the affinity of an enzyme for heparin has been exploited for its purification : lipoprotein lipase was isolated by chromatography on heparin-substituted agarose [7]. It therefore seemed worthwhile to us to explore this method for the isolation of RNA polymerase and for studying the affinity of this enzyme to matrix-bound heparin.

Affinity chromatography of salt and detergent extracts from bovine pancreas on gly-. cosylated or glycoprotein-linked Sepharose 4B resulted in purification of different carbohydratebinding proteins.Three species of proteins with molecular masses of 16 kDa, 35 kDa and 64 kDa exhibiting specificity for ß-galactosides, but none with preferential specificity for a-galactosides, were isolated from salt and detergent extracts. No Ca 2 ® was required for binding.Mannan-binding proteins of 37 kDa, 47 kDa and 94 kDa without Ca 2e -requirement were only found in the salt extract. No other mannan-binding activity could be detected. Fucose-binding proteins of 34 kDa, 62 kDa and 70 kDa exhibiting Ca 2 *-requirement for binding were present in the salt extract and two proteins with 62 kDa and 70 kDa in detergent extract. The different fractions showed agglutination activity when assayed with rabbit erythrocytes. Thus they can be defined as lectins.

Phenylalanyl-tRNA synthetases from mitochondria of yeast and hen liver resemble their corresponding cytoplasmic counterparts. Whereas slight intraspecies differences at the amino acid binding site, reflecting variations in the structures of these distinct enzymes, are exploitable by phenylalanine analogues, no intraspecies difference can be noted for the strategies to achieve the high fidelity of protein synthesis. While the yeast mitochondrial enzyme follows the pathway of posttransfer proofreading, the hen liver mitochondrial enzyme uses a tRNA-dependent pretransfer proofreading in the case of the natural amino acids. The accuracy of mitochondrial phenylalanyl-tRNA synthetases appears to be even better than the accuracy of the corresponding cytoplasmic enzymes. Interspecies rather than intraspecies differences for the functional role of certain amino acid residues of the enzymes further indicate the close relationship of the intracellular heterotopic isoenzymes. By use of a highly sensitive immunospotting procedure, common antigenic determinants are detected only within the enzymes from the two intracellular compartments of the same organism. The results suggest the origin of the cytoplasm-mitochondrion isoenzyme pair by independent gene duplication of the ancestral nuclear gene. A similarity of mitochondrial enzymes to the phenylalanyl-tRNA synthetase from Escherichia coli is not observed.

Three clones of myeloproliferative virus (MPV)-transformed rat fibroblasts (NRK) with different growth properties and morphology were transplanted to athymic nude mice. Presence of carbohydrate-binding proteins was inferred by fluorescence microscopy using fluorescent, glycosylated markers. Salt and detergent extracts of tumors from this model system were fractionated under identical conditions on different sets of Sepharose columns, to which lactose, asialofetuin, melibiose, mannan and fucose had been covalently linked. Successive elution by chelating reagent and specific sugar resulted in isolation of the different Ca2+ -dependent and Ca2+ -independent endogenous carbohydrate-binding proteins that were assayable as agglutinins. In comparison, the different tumors displayed a pattern with qualitative and quantitative alterations. Since protein-carbohydrate interaction mediated by carbohydrate-binding proteins (lectins) is of importance for cognitive processes, it is remarkable that the pattern of membrane glycoproteins, isolated by affinity chromatography on resins with immobilized plant lectins, had also been found to reveal certain individual properties for receptors specific for peanut agglutinin (PNA) and Ulex europaeus agglutinin (UEA). These demonstrated differences within the system of protein-carbohydrate interaction suggest that endogenous lectins and their ligands have potential significance as markers defining a certain phenotype within this tumor model system.

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