Affinity labelling to - SH groups in adenosine - triphosphate - phosphoribosyl transferase with the dinitrophenyl group from S-dinitrophenyl-6-mercaptopurine-riboside 5'-phosphate.

Adenosine-triphosphate-phosphoribosyl transferase from Escherichia coli reacts with S-dinitrophenyl-6-mercaptopurine-riboside 5'-phosphate. In this reaction the dinitrophenyl group becomes attached to the enzyme, while the nucleotide is split off. Most aliphatic high and low-molecular-weight-SH compounds react with the thioether in the opposite way, i.e. bind the nucleotide and split off dinitrothiophenol. It appears that the dinitrophenyl moiety of the thioether interacts with the enzyme in a specific way, and that this interaction activates the bond between the dinitrophenyl group and the sulfur atom. In support of this it was found that dinitrophenol inhibits the transferase reaction with half maximal effect at 0.4 mM. The inhibition is competitive with ATP. Dinitrophenol also competes with ATP in binding studies.     

The selective blocking of the polymerization reaction of striated muscle actin leading to a derivative suitable for crystallization. Modification of Tyr-53 by 5-diazonium-(1H)tetrazole.

The polymerization reaction of rabbit muscle actin was completely inhibited by reaction of one amino acid side chain per protein monomer with 5-diazonium-(1H)[14C]tetrazole. A tryptic peptide fingerprint showed a single peptide labeled by the reagent. The peptide was isolated and the labeled amino acid identified by amino acid analysis as Tyr-53. This side chain is not accessible to the reagent in F-actin. The modification is compared to similar inhibitions by other reagents.     

Intracellular localization of the 90 kDA heat shock protein (HSP90alpha) determined by expression of a EGFP-HSP90alpha-fusion protein in unstressed and heat stressed 3T3 cells

Heat shock protein 90 (Hsp90) is an abundant protein and essential for all eukaryotic cells. The expression of Hsp90 is further enhanced after exposure to stress factors, e.g. a heat shock. Many proteins interacting with Hsp90 as well as the various functions for Hsp90 have been described. In this study, an Hsp90alpha fusion protein along with the enhanced green fluorescence protein (EGFP) was expressed under the control of the human cytomegalovirus immediate early promoter. EGFP-Hsp90alpha was mainly localized in the cytoplasm, with only minor amounts inside the nuclei. No EGFP-Hsp90alpha could be detected inside the nucleoli. Following exposure to elevated temperatures, higher amounts of EGFP-Hsp90alpha are inside the nucleus, but not within the nucleoli. As the most remarkable finding under these conditions, an association of EGFP-Hsp90alpha with the nuclear membrane became visible.     

A strongly basic protein of the MAP2 family copolymerizes with tubulin and induces polymerization

The family of microtubuli-associated proteins of approximately 300 kD molecular weight (MAP2) from porcine brain was fractionated into components of neutral isoelectric point and one polypeptide of strongly basic nature. Both fractions are able to induce the polymerization of purified porcine brain tubulin. In the case of the fractions of an isoelectric point of 7.2, thick and short tubular structures result. Under the influence of the basic protein, extremely long tubules of normal diameter of microtubules are produced. This basic MAP2 copolymerizes with tubulin.     

Strongly basic polypeptides among microtubule associated proteins

Isoelectrofocussing between pH 3.5 and 9.5, or 9 and 11 in the first direction of two-dimensional gel electrophoreses reveal that the bands of microtubule associated proteins, e.g. the high molecular weight proteins and the τ factor, contain several polypeptides. A distinct group of these associated proteins possesses an isoelectric point of 9.5, and molecular weights between 72000 and 280000 daltons. This family of basic proteins is not the result of proteolytic cleavage of one high molecular weight precursor, as could be shown by fingerprint methods. A separation of native MAPS is briefly described.     

Mediation of anion transport in oocytes of Xenopus laevis by biosynthetically inserted band 3 protein from mouse spleen erythroid cells.

mRNA from the spleens of anemic mice was purified by oligo(dT)-cellulose chromatography and fractionated by density gradient centrifugation. After injection into oocytes of Xenopus laevis, two of the four fractions obtained led, after 16 h of incubation at 20 degrees C, to the expression of mouse band 3 protein, as demonstrated by immunoprecipitation with polyclonal antibodies against mouse band 3. Flux measurements showed an approximately 2- to 4-fold increment of 36Cl- uptake, which could be abolished by two different stilbene disulfonates, specific inhibitors of band 3-mediated anion transport in red blood cells.