Reaction of (bromoacetamido)nucleoside affinity labels with ribonuclease A: evidence for steric control of reaction specificity and alkylation rate

Four new bromoacetamido pyrimidine nucleosides have been synthesized and are affinity labels for the active site of bovine pancreatic ribonuclease A (RNase A). All bind reversibly to the enzyme and react covalently with it, resulting in inactivation. The binding constants Kb and the first-order decomposition rate constants k3 have been determined for each derivative. They are the following: 3'-(bromoacetamido)-3'-deoxyuridine, Kb = 0.062 M, k3 = 3.3 X 10(-4) s-1; 2'-(bromoacetamido)-2'-deoxyxylofuranosyluracil, Kb = 0.18 M, k3 = 1700 X 10(-4) s-1; 3'-(bromoacetamido)-3'-deoxyarabinofuranosyluracil, Kb = 0.038 M, k3 = 6.6 X 10(-4) s-1; and 3'-(bromoacetamido)-3'-deoxythymidine, Kb = 0.094 M, k3 = 2.7 X 10(-4) s-1. 3'-(Bromoacetamido)-3'-deoxyuridine reacts exclusively with the histidine-119 residue, giving 70% of a monoalkylated product substituted at N-1, 14% of a monoalkylated derivative substituted at N-3, and 16% of a dialkylated species substituted at both N-1 and N-3. Both 2'-(bromoacetamido)-2'-deoxyxylofuranosyluracil and 3'-(bromoacetamido)-3'-deoxyarabinofuranosyluracil react with absolute specificity at N-3 of the histidine-12 residue. 3'-(Bromoacetamido)-3'-deoxythymidine alkylates histidines-12 and -119. The major product formed in 57% yield is substituted at N-3 of histidine-12. A monoalkylated derivative, 8% yield, is substituted at N-1 of histidine-119. A disubstituted species is formed in 14% yield and is alkylated at both N-3 of histidine-12 and N-1 of histidine-119. A specific interaction of the "down" 2'-OH group, unique to 3'-(bromoacetamido)-3'-deoxyuridine, serves to orient the 3'-bromoacetamido residue close to the imidazole ring of histidine-119. The 2'-OH group of 3',5'-dinucleoside phosphate substrates may serve a similar role in the catalytic mechanism, allowing histidine-119 to protonate the leaving group in the transphosphorylation step. (Bromoacetamido)nucleosides are bound in the active site of RNase A in a variety of distinct conformations which are responsible for the different specificities and alkylation rates.     

DNA polymerase alpha from HeLa cells synthesizes DNA with high fidelity in a reconstituted replication system

To determine the contribution that DNA polymerase alpha makes to the overall DNA replication fidelity in mammalian systems, we measured the fidelity of replication of the SV40-based shuttle vector, pZ189, in a reconstituted in vitro DNA replication system which contained purified HeLa DNA polymerase alpha (in addition to single-stranded DNA binding protein, topoisomerase II, DNA ligase, 5'----3' exonuclease, ribonuclease H, and SV40 T-antigen). We found that DNA polymerase alpha is highly accurate when carrying out bidirectional replication in this system. This high fidelity of replication by DNA polymerase alpha in the reconstituted replication system contrasts with a relatively low fidelity of gap-filling DNA synthesis on the same target gene by purified HeLa cell DNA polymerase alpha in the absence of other replication factors. The fidelity of DNA replication by DNA polymerase alpha, although relatively high in the reconstituted system, is about 4-fold lower than DNA replication in a crude HeLa cell extract which contains additional replication factors including DNA polymerase delta. These results demonstrate that DNA polymerase alpha has the capacity to replicate DNA with high fidelity when carrying out semiconservative DNA replication in a minimal reconstituted replication system, but additional cellular factors not present in the reconstituted system may contribute to the higher replication fidelity of the crude system.     

Regulated production of recombinant echistatin by yeast

Summary The -mating factor pre-pro-leader sequence under the regulation of theGAL10 promoter was used to direct the secretion of echistatin by recombinant yeasts. Optimization of the culture medium and host strain increased the productivity of shake flask cultures twenty-fold to 8 mg/L. In fermentors, the production of echistatin was greater than 40 mg/L.     

Replication and mutagenesis of UV-damaged DNA templates in human and monkey cell extracts.

We have used in vitro DNA replication systems from human HeLa cells and monkey CV-1 cells to replicate a UV-damaged simian virus 40-based shuttle vector plasmid, pZ189. We found that replication of the plasmid was inhibited in a UV fluence-dependent manner, but even at UV fluences which caused damage to essentially all of the plasmid molecules some molecules became completely replicated. This replication was accompanied by an increase (up to 15-fold) in the frequency of mutations detected in the supF gene of the plasmid. These mutations were predominantly G:C-->A:T transitions similar to those observed in vivo. Treatment of the UV-irradiated plasmid DNA with Escherichia coli photolyase to reverse pyrimidine cyclobutane dimers (the predominant UV-induced photoproduct) before replication prevented the UV-induced inhibition of replication and reduced the frequency of mutations in supF to background levels. Therefore, the presence of pyrimidine cyclobutane dimers in the plasmid template appears to be responsible for both inhibition of replication and mutation induction. Further analysis of the replication of the UV-damaged plasmid revealed that closed circular replication products were sensitive to T4 endonuclease V (a pyrimidine cyclobutane dimer-specific endonuclease) and that this sensitivity was abolished by treatment of the replicated DNA with E. coli photolyase after replication but before T4 endonuclease treatment. These results demonstrate that these closed circular replication products contain pyrimidine cyclobutane dimers. Density labeling experiments revealed that the majority of plasmid DNA synthesized in vitro in the presence of bromodeoxyuridine triphosphate was hybrid density whether or not the plasmid was treated with UV radiation before replication; therefore, replication of UV-damaged templates appears to occur by the normal semiconservative mechanism. All of these data suggest that replication of UV-damaged templates occurs in vitro as it does in vivo and that this replication results in mutation fixation.     

Piroxicam, a structurally novel anti-inflammatory compound. Mode of prostaglandin synthesis inhibition

Piroxicam is a potent inhibitor of prostaglandin biosynthesis. Experiments utilizing cell culture and microsomes derived from various sources have demonstrated that piroxicam is a selective inhibitor of the cyclooxygenase step of arachidonic acid metabolism. Little blocking activity is observed at the phospholipase, thromboxane or prostacyclin synthetase, and arachidonic acid lipoxygenase steps.     

Modification of bovine pancreatic ribonuclease A with the site-specific reagent 4-arsono-2-nitrofluorobenzene. Spectrophotometric titration of arsononitrophenyl ribonuclease A derivatives

The 4-arsono-2-nitrophenyl chromophore can serve as a versatile spectrophotometric probe of the surface structure of proteins. Values of pK1' and pK2' for the arsonic acid ionizations are near 3 and 8, respectively, and the presence of nearby positive and negative charges produces substantial alterations in the spectral response of the probe. Changes in the extinction at the wavelength of maximum difference are 30-50% of the extinction coefficients, epsilonmax, for each ionization of the arsonic acid moiety. The titration of 41-(4-arsono-2-nitrophenyl)ribonuclease A indicates that the arsonate dianion binds near the active-site histidine residues. With protonation of a carboxylate side chain in the acidic region, presumably aspartic acid-121, the active site is disrupted. The 41-(4-arsono-2-nitrophenyl) group interacts to a greater degree with the histidine-119 side chain than it does with the histidine-12 residue. Interactions of uridine or 3'-cytidylic acid with the ligand-binding region of 41-(4-arsono-2-nitrophenyl) ribonuclease A modify the spectrophotometric response extensively. 3'-Cytidylic acid binds 41-(4-arsono-2-nitrophenyl) ribonuclease A with an affinity 300 times less than that for native ribonuclease A and 17 times lower than that for 41-(2,4-dinitrophenyl) ribonuclease A. The arsononitrophenyl chromophore is responsive to changes in the active site of ribonuclease A induced by such perturbants as ligand binding, chemical modification, and both acid and thermal denaturation.     

Alcohol-induced changes in the phospholipid molecular species of Escherichia coli.

In Escherichia coli, the additon of ethanol resulted in the synthesis of an increased proportion of phospholipids containing two unsaturated fatty acids. The addition of hexanol resulted in the opposite effect, an increase in the proportion of monounsaturated molecular species. The alcohol-induced changes were quantitatively similar to those caused by changing growth temperature. These results suggest that both adaptation to temperature and alcohol-induced changes in lipid composition share some common regulatory features.