Host Cell Enzymes Research Articles

Separation of viable Rickettsia typhi from yolk sac and L cell host components by renografin density gradient centrifugation.

Rickettsia typhi cultivated in the yolk sac of chicken embryos or in L cells irradiated 7 days previously was separated from host cell components by two cycles of Renografin density gradient centrifugation. Preliminary steps involved differential centrifugation and centrifugation over a layer of 10% bovine plasma albumin of infected yolk sac suspensions, or trypsinization and passage through filters of wide porosity of infected L cell suspensions. Rickettsial preparations obtained by these methods appeared to be free from host cell components while retaining high levels of hemolytic activity, egg infectivity, and capacity to catabolize glutamate. Average yields were 3.3 mg of rickettsial protein per yolk sac or 0.44 mg per 16-oz (ca. 475-ml) L cell culture. Extracts from these two preparations displayed malate dehydrogenase activity of electrophoretic mobility identical to each other but quite different in migration patterns from the corresponding host cell enzymes. This method of separation of rickettsiae from host cell constituents appears to be particularly well suited for the study of rickettsial enzymatic activity.

Human cytomegalovirus. III. Virus-induced DNA polymerase

Infection of WI-38 human fibroblasts with human cytomegalovirus (CMV) led to the stimulation of host cell DNA polymerase synthesis and induction of a novel virus-specific DNA polymerase. This cytomegalovirus-induced DNA polymerase was purified and separated from host cell enzymes by DEAE-cellulose and phosphocellulose column chromatographies. It can be distinguished from host cell enzymes by chromatographic behavior, template primer specificity, sedimentation property, and the requirement of salt for maximal activity. This virus-induced enzyme has a sedimentation coefficient of 9.2S and is found in both the nuclei and cytoplasm of virus-infected cells, but not in uninfected cells. This enzyme could efficiently use activated calf-thymus DNA, oly(dA)-oligo(dT)12-18, and poly(dC)-oligo(dG)12-18 as template primers, especially poly(dA)-oligo(dT)12-18, but it could not use poly(rA)-oligo(dT)12-18, poly(rC)-oligo(dG)12-18, or oligo(dT)12-18. The enzyme requires Mg2+ for maximal activity, is sensitive to p-hydroxymercuribenzoate, and is not a zinc metalloenzyme. In addition, the cytomegalovirus-induced DNA polymerase activity can be enhanced by adding 0.06 to 0.12 M NaCl or 0.03 to 0.06 M (NH4)2SO4 to the reaction mixture.

Appearance of hypoxanthine guanine phosphoribosyltransferase activity as a consequence of mycoplasma contamination.

MYCOPLASMAS are common contaminants of cells in culture and are often responsible for profound alterations in the metabolism of infected cells1,2. Such alterations include chromosomal aberrations3,4 and changes in host cell enzyme activities5. We have reported that amniotic fluid cell cultures contaminated with mycoplasmas revealed a significant increase in chromosomal aberrations over those seen in uninfected cultures6. This finding demonstrated that the presence of these organisms in amniotic fluid cell lines poses a potential hazard to prenatal chromosome diagnosis.

Separation of Viable Rickettsia typhi from Yolk Sac and L Cell Host Components by Renografin Density Gradient Centrifugation

Rickettsia typhi cultivated in the yolk sac of chicken embryos or in L cells irradiated 7 days previously was separated from host cell components by two cycles of Renografin density gradient centrifugation. Preliminary steps involved differential centrifugation and centrifugation over a layer of 10% bovine plasma albumin of infected yolk sac suspensions, or trypsinization and passage through filters of wide porosity of infected L cell suspensions. Rickettsial preparations obtained by these methods appeared to be free from host cell components while retaining high levels of hemolytic activity, egg infectivity, and capacity to catabolize glutamate. Average yields were 3.3 mg of rickettsial protein per yolk sac or 0.44 mg per 16-oz (ca. 475-ml) L cell culture. Extracts from these two preparations displayed malate dehydrogenase activity of electrophoretic mobility identical to each other but quite different in migration patterns from the corresponding host cell enzymes. This method of separation of rickettsiae from host cell constituents appears to be particularly well suited for the study of rickettsial enzymatic activity.

Phosphorylation of 5-bromodeoxycytidine in cells infected with herpes simplex virus.

5-Bromodeoxycytidine is phosphorylated to 5-bromodeoxycytidine 5'-monophosphate in extracts of cells infected with herpes simplex virus but not in extracts of uninfected cells. The conversion of 5-bromodeoxycytidine to nucleotides and its utilization for DNA synthesis in uninfected cells occurs by deamination of 5-bromodeoxycytidine to 5-bromodeoxyuridine followed by phosphorylation of 5-bromodeoxyuridine to 5-bromodeoxyuridine 5'-monophosphate. In contrast, in cells infected with herpes simplex virus, 5-bromodeoxycytidine is phosphorylated directly to 5-bromodeoxycytidine 5'-monophosphate, which can then be deaminated to 5-bromodeoxyuridine 5'-monophosphate and incorporated into DNA. These results indicate a difference in the substrate specificity of nucleoside kinases induced by herpes simplex virus and the enzymes present in uninfected cells. It is suggested that this difference in substrate specificity between virus-induced and host-cell enzymes may allow selective chemotherapy of herpes simplex infections with 5-bromo- or 5-iododeoxycytidine.

Polypeptide composition of Influenza B viruses and enzymes associated with the purified virus particles.

Influenza B/LEE/40, B/Rome/1/67, B/Hong Kong/8/73, and B/Victoria/98926/70 viruses have a similar polypeptide composition as analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. These viruses are composed of six or seven polypeptides, depending on whether one or two high-molecular-weight polypeptides are resolved, ranging in molecular weights from 27,000 to 90,400. Three of these polypeptides, namely the heavy and light hemagglutinin chains and the neuraminidase, have attached carbohydrate. Highly purified influenza B/LEE/40 and B/Rome/1/67 virus preparations have RNA-dependent RNA polymerase activity equivalent to the incorporation of 100 and 30 pmol, respectively, of (3)H-UMP per mg of virus protein per h at 37 C, which is demonstrated only in detergent-treated virus suspensions. However, no RNA-dependent DNA polymerase enzyme activity was detected in the two viruses although virus suspensions were "activated" by heat, alpha-chymotrypsin, and detergents. Other enzymatic activities were associated with purified preparations of influenza B virus and were attributed to minor contamination of virus with host cell enzymes. Thus, nucleoside and deoxynucleoside phosphohydrolase enzymes were active in the absence of detergents and catalyzed the release of 1,200 and 1,800 nmol of P(i) per mg of virus protein in 30 min at 37 C from ATP and dATP substrates. Thin-layer chromatography indicated that the products of the phosphohydrolase enzymes of influenza B/LEE/40 were mainly nucleoside diphosphate and monophosphate. The latter enzymes were tightly bound to influenza B/LEE/40 virus and could not be removed completely by repeated centrifugation, including centrifugation of the virus to equilibrium in density gradients of 25 to 40% (wt/vol) cesium chloride. A low degree of RNase (approximately 0.01 mug% contamination) and phosphatase (10-30 nmol of P(i) released per mg of virus protein per 30 min) activity was detected in some, but not all, influenza B/LEE/40 virus preparations.

Correlations between host-cell reactivation, ultraviolet reactivation and pyrimidine dimer excision in the DNA of bacteriophage λ

The relationship between the excision of pyrimidine dimers, host-cell reactivation and ultraviolet reactivation of phage λ has been investigated. From the kinetics of dimer excision and correlated biological effects the following conclusions are drawn. (1) Host-cell reactivation of phage λ involves the excision of pyrimidine dimers by host-cell enzymes. (2) Ultraviolet reactivation of λ does not involve enhancement of pyrimidine dimer excision. (3) In cells containing either irradiated λ DNA or irradiated bacterial DNA, host-cell enzymes preferentially excise dimers from host-cell DNA. By contrast, in irradiated cells infected with irradiated λ, dimers in λ DNA limit the excision of bacterial dimers better than the converse situation. These results suggest an unequal distribution of repair enzymes within the cell.

The effect of rifampin on the production of 822 phage by [formula omitted

The RNA polymerase which transcribes phage 822 following infection of Bacillus subtilis retains the rifampin sensitivity of the host cell enzyme. Competition-hybridization experiments show that several populations of phage-specific RNA molecules are produced during infection. The ability of rifampin to prevent the maturation of phage 822 is partially lost as soon as the complement of RNA species is qualitatively complete; however, the addition of the antibiotic continues to depress the yield of phage until shortly before lysis, indicating that the quantity of the late RNA species is correlated with the yield of phage.

Dependence of Vegetative Recombination Among Haemophilus influenzae Bacteriophage on the Host Cell

Vegetative recombination of temperature-sensitive mutants of Haemophilus influenzae phage HP1 cl was measured in wild-type H. influenzae strain Rd and in strain DB117, an ultraviolet-sensitive, transformation-defective mutant of the Rd strain. Recombinants are formed with low frequency in wild-type cells, but no recombination was detectable in DB117. It is concluded that these phage make use of the host cell enzymes for vegetative recombination. Lysogenization readily takes place in both strains.

Malarial parasite metabolism: The lactic acid dehydrogenase of Plasmodium berghei

The electrophoretic mobility, substrate stereospecificity, and apparent Michaelis constant ( K m ) values for lactic acid, pyruvic acid, and nicotinamide adenine dinucleotide (oxidized and reduced forms) were examined for the lactic dehydrogenase (LDH) of cell-free extracts of normal mouse erythrocytes and host cell-free P. berghei. The parasite enzyme was electrophoretically distinct from the LDH of the host cell. Both LDH's were stereospecific for l(+) lactic acid. No significant differences existed between the host cell and the parasitic LDH's in the K m values for lactic acid and nicotinamide adenine dinucleotide (oxidized and reduced forms). However, the value for pyruvate with the parasite LDH was 1 36 th that of the host cell enzyme showing an increased affinity of the former system for pyruvate. The parasitic LDH had higher specific acitivities with both lactic acid and pyruvic acid than did the host cell enzyme.