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K B Jacobson

Publications and source records attributed to K B Jacobson.

At least 55 records · Page 3Linked to original sources
PubMed1987

Partial purification of an oxygen scavenging cell membrane fraction for use in anaerobic biochemical reactions.

Anaerobic conditions are necessary to prevent the autoxidation of tetrahydrobiopterin. An enzymatic method for achieving such an anaerobic condition is shown to be obtained by a lactate oxidase activity in the membrane of Escherichia coli when the enzyme concentration exceeds 0.05 unit/ml. A procedure is described for partial purification of the membrane fragment that involves salt precipitation and gel sieve chromatography on Bio-Gel A-50m. The rate of removal of oxygen is used to define the enzyme activity, to determine stability during storage, and to define conditions for stabilization of tetrahydrobiopterin. A second assay procedure that measures the rate of reduction of resazurin is described and is useful when the enzyme concentration is low.

Anaerobiosis↗
PubMed1986

Purification of guanosine triphosphate cyclohydrolase I from Escherichia coli. The use of competitive inhibitors versus substrate as ligands in affinity chromatography.

Different affinity chromatography ligands have been compared for the purification of guanosine triphosphate (GTP) cyclohydrolase I, an enzyme that catalyses the transformation of GTP into formate and dihydroneopterin triphosphate, the first metabolite in the biosynthetic pathway of the pterins. When this enzyme is purified by affinity chromatography on GTP-Sepharose a major fraction of the activity is lost and the yield of enzyme decreases as the amount of enzyme applied to the column decreases. The use of nucleotide competitive inhibitors (UTP and ATP) as ligands in the affinity column has shown that the extent of inactivation of the enzyme is related to the affinity of the enzyme for the ligand. Further, the extent of inactivation was reduced by reducing the length of the columns when using the same volume of GTP-Sepharose. Dihydrofolate-Sepharose gave consistently higher yields of GTP cyclohydrolase I regardless of the amount of enzyme applied, but several other proteins were also obtained. For a high purification of GTP cyclohydrolase I the best yield may be obtained with UTP as the affinity ligand and with the shortest length possible of the affinity column, and the purity of enzyme is comparable with that obtained with GTP-Sepharose.

Aminohydrolases↗
PubMed1986

Nucleic acid chromatography: substitute solid support material for RPC-5 columns.

Chromatography of tRNA and DNA fragments on columns of reverse-phase 5 (RPC-5) exchange material has been widely employed for analytical and preparative studies. The Plaskon bead that formed the solid support on which a quaternary amine was absorbed is no longer commercially available. A Voltalef bead is available and provides similar, though not identical, chromatograms for Asp-tRNA, Ser-tRNA, and certain DNA fragments. Procedures are described for preparation of the column packing and for long-term operation of the column.

Chromatography, High Pressure Liquid↗
PubMed1985

Xanthurenic acid 8-O-beta-D-glucoside, a novel tryptophan metabolite in eye-color mutants of Drosophila melanogaster.

An unknown fluorescent metabolite has been isolated from heads of eye-color mutants of Drosophila melanogaster. Only a few mutations cause it to accumulate, viz. cardinal (cd), dark red brown (drb), Henna-recessive (Hnr), purple (pr), Punch2 (Pu2), Punch-Grape (PuGr), and scarlet (st). After purification by ion-exchange chromatography, the spectroscopic, chemical, and enzymatic analyses revealed that it is a novel quinoline derivative: xanthurenic acid 8-O-beta-D-glucoside. Feeding experiments suggest that this glucoside is synthesized from 3-hydroxykynurenine and that free xanthurenic acid is not a precursor. The results from the analysis for its occurrence in double mutants, together with the fact that xanthurenic acid 8-glucoside share the same precursor as xanthurenic acid and xanthommatin, suggest that xanthurenic acid 8-glucoside formation is closely related to the regulation of the last step in the biosynthesis of xanthommatin.

Animals↗
PubMed1985

Genetic and physiological parameters associated with cadmium toxicity in Drosophila melanogaster.

Two strains of Drosophila melanogaster represent the extremes in resistance and sensitivity to the lethal effects of CdCl2. The strain containing the mutations vermilion and brown (v; bw) and the strain Austin had LC50's of 3.3 and 1.3 mM CdCl2, respectively. The three major chromosomes from these two strains were assorted genetically into the six possible combinations. The measured LC50's for CdCl2 for these six genotypes fell into two groups according to the X chromosome; those containing the X chromosome from v; bw had LC50's 0.5-1.0 mM greater than those in which the X chromosome was from Austin. Since the parent strains differed by 2 mM, we suggest that the X chromosome is a major, but not the sole, site of genes to produce resistance to CdCl2. When 109Cd was in the diet the uptake by v; bw and Austin over 2 days was the same. After 4 days of uptake, the Austin strain excreted the 109Cd five times faster than v; bw but the six genotypes did not differ appreciably in excretion rate from one another and resembled the sensitive parent Austin more than the resistant one. Thus a second process is indicated that distinguishes resistance to CdCl2 that apparently is not associated with the X chromosome.

Animals↗
PubMed1984

Transfer RNA chromatography on reversed phase five: effect of cadmium ion on a queuine-type tRNA.

A sensitive method is described that detects an alteration in the structure of tRNA that is caused by cadmium but not by magnesium or zinc ions. The chromatographic system, RPC-5, separates Drosophila tyrosyl-tRNA into two fractions. These two isoacceptors differ by a single position in the anticodon where either a guanosine or queuine resides. Cadmium ions apparently interact with the tRNA and prevent the chromatographic separation. This is the first instance where cadmium is shown to cause a selective change in nucleic acid structure. The RPC-5 system seems to be uniquely useful in detecting such a change.

Animals↗
PubMed1984

Substrate and inhibitor specificity of tRNA-guanine ribosyltransferase.

We have tested as inhibitors or substrates of tRNA-guanine ribosyltransferase (EC 2.4.2.29) a number of compounds, including derivatives of 7-deazaguanine, pteridines, purines, pyrimidines and antimalarials. Virtually all purines and pteridines that are inhibitors or substrates of the rabbit reticulocyte enzyme have an amino nitrogen at the 2 position. In addition the 9 position and the oxygen at the 6 position may be important for recognition by the enzyme. Saturation of the double bond in the cyclopentenediol moiety of queuine reduces substrate activity and queuine analogs that lack the cyclopentenediol moiety, such as 7-deazaguanine and 7-aminomethyl-7-deazaguanine, are relatively poor substrates for the enzyme. While adenosine is not an inhibitor, neplanocin A (an adenosine analog in which a cyclopentenediol replaces the ribose moiety) is a poor inhibitor. The incorporation of 7-aminomethyl-7-deazaguanine into the tRNA of L-M cells results in a novel chromatographic form of tRNAAsp, indicating that L-M cells cannot modify this Q precursor (in Escherichia coli) to queuosine. The specific incorporation of 7-deazaguanine and 8-azaguanine into tRNA by L-M cells also results in novel chromatographic forms of tRNAAsp. With intact L-M cells, the enzyme-catalyzed insertion into tRNA of queuine, dihydroqueuine, 7-aminomethyl-7-deazaguanine, or 7-deazaguanine is irreversible, while guanine or 8-azaguanine incorporation is reversible; suggesting that it is the substitution of C-7 for N-7 which prevents the reversible incorporation of queuine into tRNA.

Animals↗
PubMed1984

Formation of beta,gamma-methylene-7,8-dihydroneopterin 3'-triphosphate from beta,gamma-methyleneguanosine 5'-triphosphate by GTP cyclohydrolase I of Escherichia coli.

GTP cyclohydrolase I of Escherichia coli converts [beta,gamma-methylene] GTP to a fluorescent product that is characterized as [beta,gamma-methylene]dihydroneopterin triphosphate. Interaction between the GTP analog and the enzyme gave a Ki of 3.0 microM, which may be compared to the Km of 0.1 microM for GTP. This new analog of dihydroneopterin triphosphate may, in turn, be converted to the same greenish-yellow pteridines (compounds X, X1, and X2) that are obtained from dihydroneopterin triphosphate. Because of its stability to phosphatase action, this analog may be useful for studies in pteridine metabolism.

Aminohydrolases↗
PubMed1983

Investigation of correlations between chemical parameters of metal ions and acute toxicity in mice and Drosophila.

We are studying correlations between physicochemical properties associated with metal ions and observed toxicity. In order to test correlations, we obtained, under uniform conditions, LD50 values for acute toxicity in mice for 24 metal ions. The new data show a better correlation between LD50 and Pearson and Mawby's softness parameter sigma p, defined using chemical concepts of hard and soft acids and bases, than had been obtained by others. From a wide range of physicochemical parameters, the electrode potential can give almost as good a correlation as sigma p. Better correlations might exist for parameters more relevant to biological systems.

Animals↗
PubMed1983

Toxic and biochemical effects of divalent metal ions in Drosophila: correlation to effects in mice and to chemical softness parameters.

The mechanism of toxicity of 11 divalent cations was evaluated by determining the effects of dietary administration to Drosophila melanogaster and measurement of the frequency of lethality at 4 days, alterations in the developmental patterns of proteins, and changes in specific transfer RNAs. The relative effectiveness of divalent cations to kill Drosophila is significantly correlated to the relative values of the coordinate bond energy of the metal ions. The resistance of Drosophila to cadmium toxicity appears to be genetically determined since different inbred strains vary markedly. Also, the resistance is maximal in the young adult. Two different genetic strains seem to respond to different cations (Cd2+, Hg2+, Cu2+, Co2+, Ba2+, and Sr2+) in a similar manner. Basic mechanisms of toxicity may be studied in Drosophila as well as mice since the chemical properties of the metals reflect their toxic effects on the former as closely as the latter.

Animals↗
PubMed1982

Queuine-containing isoacceptor of tyrosine tRNA in Drosophila melanogaster. Alteration of levels by divalent cations.

Dietary cadmium causes the queuine-containing, Q(+), isoacceptors to increase relative to the guanine-containing, Q(-), ones of tRNATyr, tRNAHis and tRNAAsp of Drosophila melanogaster. Of the other divalent cations examined, Sr2+, Ni2+, Cu2+, Zn2+ and Hg2+, only Hg2+ failed to cause an increase in Q(+)tRNATyr. For these results, all pre-adult stages of the organism were spent on media containing the divalent ions. Adult flies that had developed on a normal diet also responded to divalent ions; Hg2+ as well as Cd2+, Sr2+ and Zn2+ caused an increase in Q(+)tRNATyr in 4 days. Using adult flies, the rate of the response was measured; when placed on a Cd2+-containing diet, they formed significantly more Q(+)tRNATyr within 24 h as compared to adults on a normal diet. Whether the queuine is derived from the diet or from de novo synthesis is yet to be determined. Since the metal ions represent a range of values in the 'hard-soft' classification, different sites of reaction are expected, yet for Drosophila a common result is an alteration in the ratio of Q(+) and Q(-) isoacceptors of these tRNAs. The transition to Q(+)tRNA may be an early indication of the metabolic imbalances resulting from the presence of the divalent cation.

Animals↗
PubMed1982

A naturally occurring pyrimidodiazepine in Drosophila: chemical and spectral properties and relationship to drosopterin.

The structure of an intermediate, in drosopterin biosynthesis, as 6-acetylpyrimidodiazepine has been confirmed by high-resolution mass spectra, 13C NMR, chemical ionization mass spectra, and chemical properties. A trivial name of 6-acetylhomopterin is suggested and should replace the term "quench spot" used heretofore. The structure of drosopterin includes, in part, a pyrimidodiazepine, a compound that consists of a fused six- and seven-membered heterocyclic ring system. Earlier studies demonstrated that 6-acetylhomopterin strongly stimulated the enzymatic synthesis of drosopterin and related eye pigments by preparations from Drosophila. The occurrence in nature is quite limited for diazepines; drosopterin and homopterin are the first examples in eukaryotes.

Animals↗
PubMed1982

Biosynthesis, nonenzymatic synthesis, and purification of the intermediate in synthesis of sepiapterin in Drosophila.

The enzymatic conversion of the D-erythro-dihydroneopterin triphosphate [H2-neopterin-(P)3] to sepiapterin occurs via a nonphosphorylated intermediate as shown by others. We have developed a high-performance liquid chromatography assay for this intermediate and have found that the intermediate (X) and two related compounds (X1 and X2) can be formed nonenzymatically under certain conditions from H2-neopterin-(P)3. The reaction is catalyzed by tris(hydroxymethyl)aminomethane, dependent upon H2-neopterin-(P)3 concentration, significant at temperatures greater than 80 degrees C, and maximal between pH 8.5 and 9.5 (as determined at 25 degrees C). All three compounds were purified, and it was found that both X and X1 can serve as substrates for the enzymatic, NADPH-dependent synthesis of sepiapterin. From the kinetics of formation from H2-neopterin-(P)3 and the similarity of the ultraviolet spectra, it is clear that X, X1, and X2 are closely related compounds. None of the three compounds is reduced by NaBH4; only X1 is sensitive to periodate oxidation. All three can be oxidized with iodine to give rise to highly fluorescent compounds that in turn can be reduced by NaBH4 to give rise to the respective parent compounds. These latter observations indicate that X, X1, and X2 are dihydropterins. These results are discussed relative to the proposed structures for enzymatically produced X. The methods described for the nonenzymatic synthesis of X and its purification should allow preparation of large amounts of X for future study.

Drosophila↗
PubMed1982

Purification and biosynthesis of quench spot, a drosopterin precursor in Drosophila melanogaster.

Pteridine biosynthesis has been examined in extracts of the heads of Drosophila melanogaster by measuring the conversion of dihydroneopterin triphosphate to sepiapterin and the "drosopterins" (six eye pigments that are dipterin derivatives). These two products share a common first step in the production of an intermediate that is a branch point from which both products are formed. This first step can be catalyzed by sepiapterin synthase or by an enzyme found in particles that sediment at 600g. A substance named "quench spot" was found earlier to be at low levels in the purple mutants that were defective in drosopterin synthesis and to be restored to normal when a suppressor mutant, su(s)2, restored drosopterins in purple to normal levels. The sepia mutant is also deficient in the levels of both quench spot and drosopterins. In this report we propose that quench spot is a precursor of drosopterins, but not sepiapterin, and that it is formed from the sepiapterin synthase intermediate mentioned above. An additional precursor that is formed independently of the sepiapterin synthase pathway is also proposed that would react with quench spot to form drosopterins. These proposals are based on the following: (1) quench spot biosynthesis is observed in extracts of Drosophila heads in which [U-14C]dihydroneopterin triphosphate is the substrate; (2) Mg2+ is required for the synthesis of quench spot but either NADH or NADPH causes diminished incorporation of the label; (3) extracts from heads of a purple mutant (prbwcn) contain only 30% of the quench spot biosynthetic activity as compared to heads from wild type (Oregon-R); (4) quench spot has been purified from heads of wild-type Drosophila; (5) addition of quench spot stimulates the biosynthesis of drosopterins in an enzyme preparation from Oregon-R.

Animals↗
PubMed1982

Mechanism of suppression in Drosophila: evidence for a macromolecule produced by the su(s)+ locus that inhibits sepiapterin synthase.

Genetic suppression was studied in the purple mutant of Drosophila melanogaster and in suppressed purple by measurement of sepiapterin synthase activity. The addition of ammonium sulfate fractions from adult Drosophila that contain one, two, three or four doses of su(s)+ to the suppressed purple sepiapterin synthase resulted in an inhibition that increased progressively as the dosage of su(s)+ increased; the wild-type sepiapterin synthase was not inhibited. This inhibition is caused by a heat-labile macromolecule. We suggest that the mechanism of suppression is neither transcriptional nor translational but is the result of decreased amounts, or altered properties, of the normal product of the su(s)+ locus when su(s)+ is replaced by su(s)2 or su(s)e6.

Alcohol Oxidoreductases↗
PubMed1982

Dosage compensation of serine-4 transfer RNA in Drosophila melanogaster.

A dosage series of the X chromosome site for serine-4 transfer RNA consisting of one of three copies in females and one to two in males was constructed to test whether transfer RNA expression is governed by dosage compensation. A dosage effect on the level of the serine-4 isoacceptor was observed in both females and males when the structural locus was varied. However, in males, each dose had a relatively greater expression so the normal one dose was slightly greater than the total female value and the duplicated male had the highest relative expression of all the types examined. Serine-4 levels in males and females from an isogenic Oregon-R stock was similar. Thus the transfer RNA levels conform to the expectations of dosage compensation.

Animals↗