Trypsin from Greenland cod, Gadus ogac. Isolation and comparative properties

Trypsin(ogen) was isolated from the pyloric ceca of Greenland cod. Greenland cod trypsin catalyzed hydrolysis of N alpha-benzoyl-DL-arginine p-nitroanilide, tosyl arginine methyl ester and protein and was inhibited by the serine protease inhibitor PMSF and other well-known trypsin inhibitors. Greenland cod trypsin was more stable at alkaline pH than at acid pH; and was inactivated by relatively low thermal treatment. Like other trypsins, the enzyme was rich in potential acidic amino acid residues but poor in basic amino acid residues and had a molecular weight of 23,500; but it had less potential disulfide pairs, less alpha-helix and a lower H phi ave than other trypsins previously characterized. Reactions catalyzed by Greenland cod trypsin were not very responsive to temperature change, such that specific activity was relatively high at low reaction temperature.     

Protein kinase in HeLA nucleosomes: a reevaluation of the interactions of histomes with the ends of core particle DNA.

HeLa chromatin core particles contain a protein kinase which transfers phosphate from ATP to both nonhistone proteins and histones. The enzyme preferentially modifies H3 among the histones; about 7% of the H3 molecules in the nucleoprotein are modified at saturation. Activity of this kinase likely contributed to earlier results using crosslinking methodology to study which histones interact with the ends of core particle DNA. When the kinase is largely removed by sedimentation of core particles through sucrose gradients containing 0.45 M NaCl, crosslinking of the 5'-terminal label on DNA is observed only to histone H3. The overall efficiency of the crosslinking reaction is about 15%. The origin of the 5'-terminal 32P previously assigned as crosslinked to H4 is not explained by the current experiments.     

Fractionation of lymphocytes involved in the generation of cell-mediated cytotoxicity over insolubilized conjugated histamine columns.

Spleen cells from normal BALB/c mice were cultured in vitro with irradiated C57BL/6 stimulating cells. Five days later the T cell-mediated cytotoxic activity of the effector cells was assessed with a 51Cr-release assay that used H-2bEL-4 tumor cells as targets. Before the BALB/c responding lymphocytes were sensitized they were fractionated by passing the spleen cells over insolubilized histamine rabbit serum albumin Sepharose columns (H-RSA-S) or over rabbit serum albumin Sepharose (RSA-S) control columns. Fractionation of cells over the H-RSA-S columns depleted or significantly reduced the cytotoxic potential of the unretained cells. All cytotoxic potential was recovered when the cells that adhered to the H-RSA-S were eluted from the columns. In contrast, no effect on responsiveness was detected after the cells had been fractionated over the control column. The loss of response potential by the cells that did not adhere to H-RSA-S could not be accounted for by removal of macrophages nor by the concentration of cells with suppressor activity in the effluent. These cell fractionation studies raise the possiblity but do not prove that cytotoxic precursor cells may express amine receptors that could be responsible for their retention by insolubilized histamine columns.     

Comparison of various ultraviolet sources for fluorescent detection of ethidium bromide-DNA complexes in polyacrylamide gels

Ultraviolet sources with output wavelengths of 254, 300, and 366 nm were compared for detection of ethidium bromide-DNA complexes in acrylamide gels. The 254- and 300-nm sources were both much more sensitive than the 366-nm source. The 254-nm source produced a great deal of photodamage, photonicking and photodimerization, and photobleaching, while the longer wavelength sources cause little damage or bleaching. The 300-nm source is clearly the most suitable source, providing high sensitivity and a relatively low amount of photodamage and photobleaching.     

Resolution of microbial mixtures by free flow electrophoresis

Summary The resolution of bacterial mixtures by free flow electrophoresis (FFE) was not affected by the position of the microbes on the growth curve and approximately 70% of the individual cells applied were recovered as viable cells. The dependence of bacterial electrophoretic mobility on the pH, salt concentration, and viscosity of the electrolyte was determined. Suspending media and running electrolyte were developed which allowed collection of samples of>99% purity within two minutes of introduction of a mixture of Escherichia coli and Staphylococcus aureus. Most bacterial strains migrated in a single band, although some migrated in more than one band. Escherichia coli was resolved from each of 10 different species. The considerable variation in mobility found in 21 different E. coli strains, however, appears to preclude use of FFE as a method of species identification.     

Properties of a gonococcal inhibitor produced by Escherichia coli.

Strains of Escherichia coli can inhibit the in vitro growth of Neisseria gonorrhoeae. One E. coli strain released a potent agar-diffusible gonococcal growth inhibitor which was extracted and assayed in an agar well assay system. The culture conditions necessary to produce the inhibitor were determined. The inhibitor was bacteriostatic, in most cases, for N. gonorrhoeae. Based on ultrafiltration and column chromatography, the inhibitor appeared to have a molecular weight in the range of 1200 to 2000. Evidence that the molecule contained charged sites was obtained by membrane binding and column chromatography. The inhibitor was stable to extremes of heat, cold and pH. It was not volatile or susceptible to proteolytic enzymes, lysozyme, lipase, DNAase, RNAase or certain chelating agents. Its activity was completely blocked by ferric ammonium citrate. This inhibitor is dissimilar to previously reported gonococcal inhibitors of bacterial origin.     

Galactolipid Synthesis in Vicia faba Leaves: IV. Site(s) of Fatty Acid Incorporation into the Major Glycerolipids

The fatty acids of the major glycerolipids of Vicia faba leaves were analyzed immediately following ¹⁴CO₂ feeding. The leaves were fractionated into chloroplast and cytoplasmic fractions and the location of radioactivity in the fatty acids determined. The results indicate that the major site of incorporation of fatty acids is in the phospholipids. Phosphatidylcholine contained the highest level of radioactivity in the cytoplasmic fraction, whereas phosphatidylglycerol contained radioactivity in both the chloroplast and cytoplasmic fractions. The galactolipids contained very little radioactivity in comparison, this radioactivity being confined to high speed centrifugal fractions believed to contain the envelopes of the chloroplast. Our results suggest that phosphatidylcholine is a major site of incorporation of fatty acids (mainly in oleic acid) in the cytoplasm, whereas phosphatidylglycerol is also a site of incorporation involving both oleic and palmitic acids, inside and outside the chloroplast.