Crystallization of trimeric recombinant human tumor necrosis factor (cachectin)

Crystals of tumor necrosis factor (TNF) have been obtained in two forms. Rhombohedral crystals grow in 1.8 to 2.0 M ammonium sulfite, pH 7.8 at 21 degrees C, and tetragonal crystals grow in 2.6 M magnesium sulfate, pH 5.5 at 25 degrees C. Analysis of TNF by isoelectric focusing under native and denaturing conditions indicates that TNF molecules exist as trimers in solution. The rhombohedral cachectin crystals belong to space group R3 and have unit cell constants a = b = c = 47.65 A and alpha = beta = gamma = 88.1 degrees. Density determinations and the space group indicate that the unit cell contains one 51,000-dalton trimer. These crystals are stable in the x-ray beam and diffract to at least 1.85 A but are apparently twinned by merohedry. The tetragonal crystals are space group P4(3)2(1)2 or its enantiomorph P4(1)2(1)2 and have unit cell constants a = b = 95.08, c = 117.49. The asymmetric unit contains one trimer; the crystals are stable in the x-ray beam and diffract to beyond 3 A.     

Intermolecular histone H4 interactions in core nucleosomes

Chicken histone H4, labeled at methionine-84 with 1-N-pyrenyliodoacetamide, has been incorporated into a nucleosome-like particle with core length DNA and unmodified histones H2A, H2B, and H3. These synthetic nucleosomes exhibit properties very similar to those displayed by native particles and those labeled with other fluors. The emission spectrum of the pyrene-labeled nucleosome was characteristic of excited dimer (excimer) fluorescence, indicating that the single pyrene groups on the two H4 molecules are in close proximity in the reconstituted particle. Histone H4 was also labeled randomly at lysines with a group that contains two pyrene moieties separated by 12 A at most. Incorporation of this histone into nucleosome-like particles provides an excimer standard which does not depend on intermolecular interactions. The properties of the pyrene-containing nucleosome were examined as a function of ionic strength. It was found that the H4-H4 pyrene excimer fluorescence exhibited a cooperative disruption centered at 0.1 M NaCl which preceded increases in accessibility and environment polarity revealed by other fluors attached at the same site.     

Loss of sigma-factor of RNA polymerase of Xanthomonas campestris pv. oryzae during phage Xp10 infection

Ten min after infection of Xanthomonas campestris pv. oryzae by phage Xp10, a sharp decrease in the activity of the host RNA polymerase was observed. Host RNA polymerase from phage-infected and uninfected cells was purified, and their properties were compared. The enzyme from uninfected cells contained four polypeptides with Mr = 155,000, 155,000, 93,000, and 37,000, respectively, and assembled with a stoichiometry of alpha 2 beta beta' sigma. The enzyme from infected cells lacked the sigma-subunit. The enzyme from uninfected cells utilized Xp10 DNA and poly[d(A-T)] as templates, the enzyme from phage-infected cells failed to transcribe Xp10 DNA, but retained the ability to transcribe poly(A-T). The regions of the Xp10 genome transcribed by the two enzymes were also investigated. The enzyme from uninfected cells transcribed the leftmost 25-30% of the Xp10 genome. The enzyme from phage-infected cells also transcribed the same region, but the enzyme activity was very low. Other properties such as (a) the response to RNA polymerase inhibitors, (b) the effect of N-ethylmaleimide, (c) the requirement of Mg2+ and Mn2+, and (d) the optimum temperature and pH of the two enzymes were very similar.     

DNA amplification in multidrug, cross-resistant Chinese hamster ovary cells: molecular characterization and cytogenetic localization of the amplified DNA

Vincristine-resistant (VCR) Chinese hamster ovary (CHO) cells have been established by stepwise selection in increasing concentrations of vincristine. These cells exhibit multidrug cross-resistance to a number of drugs that have no structural or functional similarities. Cytogenetic analyses of resistant cells revealed the presence of double minutes and expanded chromosomal segments, thus implicating gene amplification as a possible mechanism of resistance. An amplified DNA segment isolated from other multidrug cross-resistant CHO cell lines (Roninson, I. B., H. T. Abelson, D. E. Housman, N. Howell, and A. Varshavsky, 1984, Nature (Lond.), 309:626-628) is also amplified in our VCR lines. This DNA segment was used as a probe to screen a cosmid library of VCR genomic DNA, and overlapping clones were retrieved. All of these segments, totaling approximately 45 kilobases (kb), were amplified in VCR cells. Using in situ hybridization, we localized the amplification domain to the long arm of CHO chromosome 1 or Z1. Northern hybridization analysis revealed that a 4.3-kb mRNA was encoded by this amplified DNA domain and was over-produced in the VCR cells. Suggestions for the involvement of these amplified DNA segments in the acquisition of multidrug cross-resistance in animal cells are also presented.     

Substrate specificity and protonation state of ornithine transcarbamoylase as determined by pH studies

The ornithine transcarbamoylase catalyzed reaction and its inhibition by L-norvaline have been investigated between pH 5.5 and 10.5. The steady-state turnover rate (kcat) of the enzyme from Escherichia coli increases with pH and plateaus above pH 9. Its change with pH conforms to a single protonation process with an apparent pKa of 7.3. The effect of pH on the apparent Michaelis constant (KMapp) of L-ornithine suggests that this diamino acid in its cationic form is not the substrate. Treating only the zwitterions of ornithine as substrate, the pH profile of the pseudo-first-order rate constant (kcat/KMz) of the reaction is a bell-shaped curve characterized by pKa's of 6.2 and 9.1 and asymptotic slopes of +/- 1. Similar pKa's (6.3 and 9.3) are obtained for the pKi profile of zwitterionic L-norvaline, a competitive inhibitor. The pKi profile further indicates that the alpha-amino group of the inhibitor must be charged for binding. Together, these pH profiles provide sufficient information to suggest that only the minor zwitterionic species of ornithine, H2N(CH2)3CH(NH3+)COO-, binds the enzyme productively. The selection of this substrate form by the enzyme leads to a Michaelis complex in which ornithine is poised for nucleophilic attack. Following such binding, the need for deprotonation of the delta-NH3+ group is avoided, and transcarbamoylation becomes energetically more feasible. Reaction schemes accounting for the effects of pH are proposed for the enzymic reaction.     

A specific iron stain for iron-binding proteins in polyacrylamide gels: application to transferrin and lactoferrin

A new method for specifically staining the iron atoms present in transferrin and lactoferrin after polyacrylamide gel electrophoresis and isoelectric focusing is described. The stain, 3-(2-pyridyl)-5,6-bis(2-(5-furylsulfonic acid))-1,2,4-triazine, disodium salt, or Ferene S, will detect transferrin in 5 microliters of human serum, lactoferrin in 10 microliters of human whey, and 10 micrograms of purified primate (Macaca fascicularis) transferrin. This method of staining is very rapid as the serum transferrin bands can be seen within 5 to 10 min of staining.     

Monoclonal antibodies to cytochrome c from Paracoccus denitrificans: effects on electron transport reactions

The effect of a monoclonal antibody to a soluble cytochrome c from Paracoccus denitrificans was tested on the membrane-bound electron-transport system of this bacterium. This antibody (F3-10.2) and one previously described (F3-29.4) (Kuo, L.M., Davies, H.C. and Smith, L. (1984) Biochim. Biophys. Acta 766, 472-482) were deduced to bind to the cytochrome c in the area including amino acid residue number 23 on a loop on the side of the heme crevice. In contrast to the observations with the previously tested antibody, the present data show the second antibody to block completely the reaction of the cytochrome c with cytochrome c oxidase but not that with cytochrome c reductase. Neither antibody has an appreciable inhibitory effect on the NADH oxidase of the isolated detergent-treated membranes. The two antibodies bind in different ways, giving insight into the interaction of a soluble protein with membrane-bound enzymes. The data indicate that the reaction sites on the cytochrome c for the oxidase and reductase moieties of P. denitrificans are different. They also argue against the need for a dissociable cytochrome c comparable to that which functions on the mitochondrial inner membrane.     

Deoxycholate induces the preferential hydrolysis of polyphosphoinositides by human platelet and rat corneal phospholipase C

Deoxycholate promotes phospholipase C degradation of endogenous phosphatidyl[3H]inositol (Pl), phosphatidyl[3H]inositol monophosphate (PIP) and phosphatidyl[3H]inositol bisphosphate (PIP2) in rat cornea and human platelets. Hydrolysis of phosphatidyl[3H]inositol significantly lags polyphospho[3H]inositide degradation. Concomitantly, formation of [3H]inositol monophosphate (IP1) lags behind [3H]inositol bisphosphate (IP2) and [3H]inositol trisphosphate (IP3) production. These results demonstrate that rat cornea and human platelet phospholipase C cause a preferential hydrolysis of the endogenous polyphosphoinositides rather than phosphatidylinositol.