Purification and characterization of uracil phosphoribosyltransferase from Crithidia luciliae

1. Uracil phosphoribosyltransferase (UPRTase) was purified 370-fold from the protozoan parasite, Crithidia luciliae. 2. The enzyme was a dimer of mol. wt 80 000 and was highly specific for uracil. 3. GTP, which is an activator of UPRTase from E. coli had a slight inhibitory effect on the parasite enzyme. 4. The C. luciliae UPRTase demonstrated a broad specificity for activating divalent metal ions.     

Gender differences in anaerobic power tests

The purpose of this study was to determine if the differences in anaerobic power between males and females could be accounted for by differences in body composition, strength, and neuromuscular function. A total of 82 untrained men and 99 women took part in the study. Body composition, somatotype, isometric strength, neuromuscular function were measured, and four anaerobic power tests performed. The men were significantly different from the women on all strength, power, and neuromuscular measurements except reaction time and on all anthropometric and somatotype dimensions except ectomorphy. Strength and anthropometric dimensions were similarly related to anaerobic power values within each sex. Relative fat (%fat) exerted different degrees of influence on sprint and jump performances in each sex. Removing the influence of anthropometric, strength, and neuromuscular differences by analysis of covariance reduced, but did not remove, the significant differences between the sexes. Therefore, factors other than lean body mass, leg strength, and neuromuscular function may be operating in short-term, explosive power performances to account for the differences between the sexes. The task-specific nature of anaerobic power tests and the relatively large influence of anthropometric factors on power production were confirmed.     

Purification and spectral study of a microbial fatty acyltransferase: activation by limited proteolysis

A fatty acyltransferase with a reaction mechanism similar to that of mammalian lecithin: cholesterol acyltransferase has been purified from culture supernatants of a mutant Aeromonas salmonicida containing the cloned Aeromonas hydrophila structural gene. Typically, more than 35 mg of protein were isolated from 2 L of culture supernatant. The amino-terminal sequence, amino acid composition, and molecular weight of the purified protein corresponded to predictions based on the sequence of the gene, indicating that the signal sequence had been correctly removed during export but that no further processing had occurred. Analysis of the far-UV circular dichroic (CD) spectrum of the enzyme showed that it consists of 31% alpha-helix, 21% beta-sheet, and 16% beta-turn, with 12% of aperiodic form. Treatment of the purified protein with a variety of proteases resulted in nicking near the C-terminus. This led to an increase in enzyme activity against lipids in erythrocyte membranes and increased rate of hydrolysis of p-nitrophenyl butyrate. Activation was accompanied by a change in the CD spectrum and a change in its aggregation state. The trypsin cut site was located between the two cysteines in the enzyme. Evidence is presented that the cysteines are joined by a disulfide bond and therefore cannot participate in acyl transfer. This may distinguish the microbial enzyme from lecithin:cholesterol acyltransferase. This is the second extracellular A. hydrophila protein that we have shown can be activated by proteolysis after it is released.     

Characterization of the deoxyribonuclease activity of diphtheria toxin

Having discovered that the A domain of diphtheria toxin exhibits intrinsic nuclease activity (Chang, M. P., Baldwin, R. L., Bruce, B., and Wisnieski, B. J. (1989) Science 246, 1165-1168), we proceeded to examine the requirements for optimal enzymic expression. In vitro assays with linear double-stranded DNA demonstrated that optimal activity occurs at pH 7.5 and 37 degrees C. A characterization of the stringent cation-dependence of the reaction revealed increasing activity with increasing Mn2+ up to 30 mM. In contrast, activity levels with Ca2+ or Zn2+ alone peaked at 100 microM and with Mg2+ alone at 1 mM. The Zn2(+)- and Mg2(+)-stimulated activities appear to be dependent on trace amounts of Ca2+. Indeed, inclusion of 2 mM Ca2+ plus 3 mM Mg2+ in the reaction buffer promoted a high level of DNA cleavage even though very little cleavage was seen with either cation alone at 2-3 mM. Addition of 20-200 mM NaCl or KCl caused progressive inhibition. Detection of diphtheria toxin nuclease activity under physiologically relevant conditions suggests that it may be operative in vivo and supports our contention that diphtheria toxin-induced cytolysis is not a simple consequence of protein synthesis inhibition, but rather the final step in a cytolytic pathway linked to chromosomal integrity.     

Plasmid transformation and replica filter plating of Acholeplasma laidlawii

The restriction deficient mutant 8195 of Acholeplasma laidlawii strain JA1 was transformed by the promiscuous streptococcal plasmid vector pNZ18 at a frequency of 4 x 10(-4)/cfu. The plasmid was maintained without structural rearrangements but was lost in the absence of a selection pressure, i.e. kanamycin or neomycin. Transformed primary colonies were easily recognized due to a different colony morphology. Replica filter plating, previously not obtained with mycoplasmas, was achieved using pNZ18 as a marker by incubating the replica filters with the cell side down on the new agar plates. These findings should greatly facilitate the genetic and functional analysis of A. laidlawii.     

Microbody phosphoglycerate kinase of Trypanosoma brucei: expression and complementation in Escherichia coli

In the primitive eukaryotic parasite, Trypanosoma brucei, most of the enzymes of glycolysis are located within microbody organelles called glycosomes. Proteins destined for the glycosome are synthesized on free ribosomes and post-translationally translocated into the organelle. The gene, gPGK, encoding the glycosomal isozyme of phosphoglycerate kinase (gPGK), was cloned adjacent to a T7 promoter and cotransformed with a plasmid encoding T7 RNA polymerase into Escherichia coli Pgk-cells. Functional complementation occurred, but only after the creation of a ribosome-binding site by mutagenesis. This represents the first example of complementation of an E. coli mutant with a gene encoding a microbody protein. Enzymatically active recombinant gPGK was purified to near homogeneity by ion exchange chromatography from highly expressing E. coli. The recombinant protein will aid in studies of glycosomal biogenesis.     

Changes in Ca, Mg-dependent endonuclease of DNA in isolated nuclei of human lymphocytes in lymphoproliferative diseases

Ca, Mg-dependent endonuclease is one of the main DNAses of lymphocyte chromatin. It's activity is known to increase in the immune response and to decrease in spontaneous and experimental CLL. These observations became a basis for analysis of possible clinical meaning of it's enzymatic activity assay. Donors' peripheral blood lymphocytes being tested, normal level of endonucleolysis for men and children was defined. Except that patients with different clinical forms of lymphoproliferative diseases such as chronic lympholeukemia, non-Hodgkin lymphomas, Hodgkin's disease were observed. The results showed that Ca, Mg-dependent endonucleolysis activity was decreased in comparison to donors' one. Ca, Mg-dependent endonucleolysis activity was the same in the group of patients with non-malignant pathology and in donors' one. Successful treatment and remission state of our patients was associated with alteration of the Ca, Mg-dependent endonucleolysis activity to normal level as well as immunological parameters. That is why the activity of Ca, Mg-dependent endonucleolysis is suggested to be a new criterion of immune state and lymphocyte malignant transformation.