Development of rational methods for purifying restriction endonucleases

The work deals with the search for rational schemes of the purification of endonucleases, suitable for the isolation of different enzymes. The scheme using the combination of Blue Sepharose and phosphocellulose has proved to be most universal. The expediency of starting the purification of Haemophilus enzymes on biogel A-0.5m has been established.     

Xyloside transport by XylP, a member of the galactoside-pentoside-hexuronide family.

This paper describes the functional characterization of the xyloside transporter, XylP, of Lactobacillus pentosus with the aid of a spectroscopy-based assay system. In order to monitor the transport reaction, the natural xyloside isoprimeverose, a building block of hemicellulose, and the analogue methyl-isoprimeverose were chemically synthesized by a new and efficient procedure. The XylP protein was purified by metal affinity chromatography, following high level expression in Lactococcus lactis from the nisin-inducible promoter. The purified XylP protein was incorporated into liposomes, in which the glucose dehydrogenase from Acinetobacter calcoaceticus (sGDH) was entrapped. sGDH can oxidize aldose sugars in the presence of dichlorophenol-indophenol as electron acceptor. The coupled assay thus involves XylP-mediated isoprimeverose uptake followed by internal oxidation of the sugar by sGDH, which can be monitored from the reduction of 2,6-dichlorophenol-indophenol at 600 nm. The uptake of isoprimeverose was stimulated by the presence of the non-oxidizable methyl-isoprimeverose on the trans-side of the membrane, indicating that exchange transport is faster than unidirectional downhill uptake. Unlike other members of the galactoside-pentoside-hexuronide family, XylP does not transport monosaccharides (xylose) but requires a glycosidic linkage at the anomeric carbon position. Consistent with a proton motive force-driven mechanism, the uptake was stimulated by a membrane potential (inside negative relative to outside) and inhibited by a pH gradient (inside acidic relative to outside). The advantages of the here-described transport assay for studies of carbohydrate transport are discussed.     

Effect of hormonal and neurotrophic factors on the expression of fast myosin in slow muscle

Myosin ATPase and succinic dehydrogenase activity and fast myosin (indirect immunochemical test) were assayed in m. soleus of guinea-pigs after the administration of T4 (200 micrograms/kg) to animals every day for 3 weeks. This was followed by the application of 10 mM colchicine solution to sciatic nerve for 6 min. Fast muscle fibers and the line of precipitation with antiserum to fast myosin were revealed in soleus muscle of experimental animals after the application of colchicine and T4 injection.     

Trypanosoma rhodesiense blood forms express all antigen specificities relevant to protection against metacyclic (insect form) challenge

Monoclonal antibodies were used to demonstrate the expression of four distinct metacyclic (infective insect form) trypanosome antigens on blood forms of T. rhodesiense. Metacyclic antigens were consistently expressed on the blood forms on days 4 and 5 of the first parasitemia after metacyclic infection of C57BL/6 mice. In different mice examined, the percent of blood forms expressing metacyclic antigens ranged from 46 to 85%. Immunization with irradiated day-5 blood form trypanosomes was protective against metacyclic challenge, indicating that all antigen specificities relevant to protective immunization against metacyclic challenge are expressed on blood form trypanosomes. Blood forms, in contrast to metacyclic forms, can be isolated in quantities sufficient for purification of antigens and genetic cloning.     

An assay for the detection of specific binding of 3-methylcholanthrene to rat liver cytosolic proteins using DEAE-cellulose

A method for the detection of the specific binding of 3-methylcholanthrene to rat liver cytosolic proteins is described. The separation of the protein-bound 3-methylcholanthrene from the free 3-methylcholanthrene was achieved using a batch DEAE-cellulose technique. Extraction of the DEAE-cellulose with 0.3 M KCl allowed the selective release and measurement of the amount of protein-bound 3-methylcholanthrene. The assay was optimized for the following parameters: time of incubation with DEAE-cellulose, time required for salt extraction, protein concentration, the concentration of KCl required to elute the specific binding proteins, the amount of DEAE-cellulose required to bind the specific binding proteins, and ligand specificity. The sedimentation properties of those 3-methylcholanthrene-binding proteins which were extracted with salt from DEAE-cellulose were examined on 5 to 20% sucrose gradients; the major binding species sedimented as a broad peak at 4.5 S.