Active site mutations in DNA topoisomerase I distinguish the cytotoxic activities of camptothecin and the indolocarbazole, rebeccamycin.

DNA topoisomerase I (Top1p) catalyzes topological changes in DNA and is the cellular target of the antitumor agent camptothecin (CPT). Non-CPT drugs that target Top1p, such as indolocarbazoles, are under clinical development. However, whether the cytotoxicity of indolocarbazoles derives from Top1p poisoning remains unclear. To further investigate indolocarbazole mechanism, rebeccamycin R-3 activity was examined in vitro and in yeast. Using a series of Top1p mutants, where substitution of residues around the active site tyrosine has well-defined effects on enzyme catalysis, we show that catalytically active, CPT-resistant enzymes remain sensitive to R-3. This indolocarbazole did not inhibit yeast Top1p activity, yet was effective in stabilizing Top1p-DNA complexes. Similar results were obtained with human Top1p, when Ser or His were substituted for Asn-722. The mutations altered enzyme function and sensitivity to CPT, yet R-3 poisoning of Top1p was unaffected. Moreover, top1delta, rad52delta yeast cells expressing human Top1p, but not catalytically inactive Top1Y723Fp, were sensitive to R-3. These data support hTop1p as the cellular target of R-3 and indicate that distinct drug-enzyme interactions at the active site are required for efficient poisoning by R-3 or CPT. Furthermore, resistance to one poison may potentiate cell sensitivity to structurally distinct compounds that also target Top1p.     

Biochemical characterization of recombinant human phenylalanine hydroxylase produced in Escherichia coli

A full-length human phenylalanine hydroxylase cDNA has been recombined with a prokaryotic expression vector and introduced into Escherichia coli. Transformed bacteria express phenylalanine hydroxylase immunoreactive protein and pterin-dependent conversion of phenylalanine to tyrosine. Recombinant human phenylalanine hydroxylase produced in E. coli has been partially purified, and biochemical studies have been performed comparing the activity and kinetics of the recombinant enzyme with native phenylalanine hydroxylase from human liver. The optimal reaction conditions, kinetic constants, and sensitivity to inhibition by aromatic amino acids are the same for recombinant phenylalanine hydroxylase and native phenylalanine hydroxylase. These data indicate that the recombinant human phenylalanine hydroxylase is an authentic and complete phenylalanine hydroxylase enzyme and that the characteristic aspects of phenylalanine hydroxylase enzymatic activity are determined by a single gene product and can be constituted in the absence of any specific accessory functions of the eukaryotic cell. The availability of recombinant human phenylalanine hydroxylase produced in E. coli will expedite physical and chemical characterization of human phenylalanine hydroxylase which has been hindered in the past by inavailability of the native enzyme for study.     

Dendritic cell activating factor produced by mouse macrophage hybridoma

A mouse macrophage (M phi) hybridoma which produces a soluble factor responsible for the cooperation between M phi and spleen dendritic cells (DC) was constructed. The antigen-presenting activity and the stimulator cell activity in the allogeneic or syngeneic mixed leukocyte reaction of DC were significantly augmented when DC were incubated with the culture supernatant of the hybridoma treated with various stimulants including latex beads. The monokine present in the culture supernatant of the hybridoma, called dendritic cell-activating factor (DCAF), augmented the production of lymphocyte-activating factor by DC while Ia expression of DCAF-treated DC was not altered. DCAF had no effect on the antigen-presenting activity of peritoneal resident M phi or B cell blasts while the antigen-presenting activity of spleen M phi was enhanced, but the degree of the enhancement was much less than that of spleen DC. DCAF was found to have the following properties: its pI value is between pH 4 and 5; it is stable at pH 2 to 10; and it loses its activity on incubation at 75 C for 30 min. When the culture supernatant of the hybridoma stimulated with latex beads was subjected to gel filtration, the DCAF activity was detected in the 20 Kd to 25 Kd, 30 Kd to 40 Kd, and 50 Kd to 60 Kd molecular weight regions. The 30 Kd to 40 Kd fraction, which is the major peak fraction, was further purified by ion-exchange chromatography followed by gel-filtration chromatography. When each fraction was subjected to SDS-PAGE, a 30 Kd band corresponding to the DCAF activity was observed and DCAF was purified to about 90% purity.     

Pigment types in sheep, goats, and llamas

Pigment types in various colors of fiber from sheep, goats, and llamas were assayed by a method using high performance liquid chromatography. In these three species the black/gray group is due to eumelanin, which is fully intense in all three species. Red phenotypes are due to pheomelanin and fade considerably with age in fiber from sheep and goats, but not in llamas. This phenomenon has implications on the genetic mechanisms used in generating white fiber. Brown phenotypes in sheep are due to eumelanin, in goats these phenotypes are equivocal, and they were not observed in llamas.     

Pigment types of various color genotypes of horses

Hair samples of various colors of horses were analyzed for content of both eumelanin and pheomelanin by a procedure using high performance liquid chromatography. The results are in accord with generally accepted genetic hypotheses accounting for the various colors. However, the results support the hypothesis that the chestnut/sorrel group of colors is conditioned by the extension locus, not the brown locus. The results also indicate that the brown locus is a likely contributor to some rare color phenotypes.     

Site-site interactions in EF-hand calcium-binding proteins. Laser-excited europium luminescence studies of 9-kDa calbindin, the pig intestinal calcium-binding protein

Europium(III) binding to 9-kDa calbindin from pig intestines was studied by direct excitation of the 7Fo----5Do transition of the ion and by near-ultraviolet circular dichroic spectroscopy. Europium(III) binding is clearly biphasic. As with other lanthanides the C-terminal metal-binding site (site II) is filled first. The europium ion in this site gives an excitation spectrum with a single peak at 579.1 nm (peak 2). The occupation of the N-terminal site (site I) by europium gives excitation spectra that are pH-dependent and show a peak at 579.4 nm (peak 1a) at pH 5 which shifts to 578.7 nm (peak 1b) over the pH range 5-7. At pH 8.07 the fluorescence from europium in site I largely disappears because of weak binding, whereas that from site II is quenched by about 75% in spite of full occupancy of the site as shown by circular dichroic titration. There is a strong interaction between the two sites in spite of the very different affinities. The fluorescence from site II increases stoichiometrically with the addition not only of the first equivalent of europium, but also concomitantly with the fluorescence from site I upon addition of the second equivalent. Furthermore, when Eu1-calbindin is titrated with calcium the fluorescence at 579.1 nm is quenched by about 30% during the addition of one equivalent of calcium which fills site I. Subsequent titration with large excesses of calcium displaces europium from site II. The affinity of site II for europium is about 100 times that of calcium under these conditions.     

Identification by molecular cloning of two forms of the alpha-subunit of the human liver stimulatory (GS) regulatory component of adenylyl cyclase

Two DNA molecules complementary to human liver mRNA coding for the alpha-subunit of the stimulatory regulatory component Gs of adenylyl cyclase were cloned. One of the two forms is a full-length cDNA of 1614 nucleotides plus a poly(A) tail of 59 nucleotides. The deduced sequence of 394 amino acids encoded by its open reading frame is essentially identical to that of the alpha-subunits of Gs identified by molecular cloning from bovine adrenals, bovine brain and rat brain. Two independent clones of the other type of cDNA were isolated. Both were incomplete, beginning within the open reading frame coding for the alpha s polypeptide. One codes for amino acids 5 through 394 and the other for amino acids 48 through 394 of the above described cDNA of 1614 nucleotides, and both have the identical 3'-untranslated sequence. They differ from the first cDNA, however, in that they lack a stretch of 42 nucleotides (numbers 214 through 255) and have nucleotides 213 (G) and 256 (G) replaced with C and A, respectively. This results in a predicted amino acid composition of another alpha-subunit of Gs that is shorter by 14 amino acids and contains two substitutions (Asp for Glu and Ser for Gly) at the interface between the deletion and the unchanged sequence. We call the smaller subunit alpha s1 and the larger alpha s2. This is the first demonstration of a structural heterogeneity in alpha s subunits that is due to a difference in amino acid sequence.     

Comparison of crude and affinity purified cytosolic epoxide hydrolases from hepatic tissue of control and clofibrate-fed mice

An affinity purification procedure was developed for the cytosolic epoxide hydrolase based upon the selective binding of the enzyme to immobilized methoxycitronellyl thiol. Several elution systems were examined, but the most successful system employed selective elution with a chalcone oxide. This affinity system allowed the purification of the cytosolic epoxide hydrolase activity from livers of both control and clofibrate-fed mice. A variety of biochemical techniques including pH dependence, substrate preference, kinetics, inhibition, amino acid analysis, peptide mapping, Western blotting, analytical isoelectric focusing, and gel permeation chromatography failed to distinguish between the enzymes purified from control and clofibrate-fed animals. The quantitative removal of the cytosolic epoxide hydrolase acting on trans-stilbene oxide from 100,000g supernatants, allowed analysis of remaining activities acting differentially on cis-stilbene oxide and benzo[a]pyrene 4,5-oxide. Such analysis indicated the existence of a novel epoxide hydrolase activity in the cytosol of mouse liver preparations.