PubMed Health⌕ Search

Biomedical subjects

W R Farkas

Publications and source records attributed to W R Farkas.

At least 37 records · Page 2Linked to original sources
PubMed1983

Partial purification and characterization of dihydrodipicolinic Acid reductase from maize.

Dihydrodipicolinic acid reductase, an enzyme which catalyzes the pyridine nucleotide-linked reduction of dihydrodipicolinic acid to tetrahydrodipicolinic acid in the biosynthetic pathway leading to l-lysine, has been partially purified from maize (Zea mays cv Pioneer 3145) kernels. The crude maize extract and the partially purified enzyme were assayed for dihydrodipicolinic acid reductase by their ability to restore the capability of crude extracts of a mutant Escherichia coli (CGSC 4549; defective in dihydrodipicolinic acid reductase) to synthesize diaminopimelic acid from aspartic acid and pyruvic acid.In a study of its properties, the Michaelis constant obtained for dihydrodipicolinic acid was 4.3 x 10(-4) and for NADPH the K(m) was 4.6 x 10(-5). The enzyme had a pH optimum close to 7 and was much more temperature labile than the bacterial enzyme. Its molecular weight was 8.0 x 10(4).Several compounds, viz., alpha-picolinic acid, l-pipecolic acid, isophthalic acid, and isocinchomeronic acid, with structures similar to dihydrodipicolinic acid inhibited the reductase reaction. Dipicolinic acid, the most potent of these inhibitors, acted as a competitive inhibitor with a K(i) of 9.0 x 10(-4). The competitive inhibition of the reductase reaction by dipicolinic acid (oxidized dihydrodipicolinic acid) suggests that the substrate for this enzyme was in the ring rather than the open chain form. Oxidized pyridine nucleotide inhibited the activity slightly.

Journal Article↗
PubMed1982

Purification and properties of guanine, queuine-tRNA transglycosylase from wheat germ.

Guanine, queuine-tRNA transglycosylase has been purified from wheat germ to homogeneity. The specific activity is 2,000 pmol h-1 mg-1 of protein. The enzyme has an apparent Mr = 140,000. It migrates as a single band with Mr = 68,000 on electrophoresis in sodium dodecyl sulfate gels, indicating two Mr = 68,000 subunits. Both guanine (Km = 6.0 X 10(-8) M) and queuine (KI = 9.5 X 10(-8) M) are substrates but 7-(aminomethyl)-7-deazaguanine is not. The enzyme requires a monovalent or a divalent cation; Na+ and Mg2+ are more effective activators than other cations. The optimum pH is 7.6. Six tRNA isoacceptors found in wheat germ are substrates for the enzyme.

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↗
PubMed1981

Administration of exogenous queuine is essential for the biosynthesis of the queuosine-containing transfer RNAs in the mouse.

1. Normal mouse liver contains predominantly tRNA that contains queuosine in the first position of the anticodon ((Q+)tRNA). 2. Germ-free mice fed a chemically defined diet devoid of queuine for 1 year have no queuine in all four of the tRNAs that respond to the NAUC codons. 3. The synthesis of (Q+)tRNAs can be induced by injecting queuine, feeding free queuine, or by feeding (Q+)tRNA. 4. When mice that have no (Q+)tRNA are titrated with exogenous queuine, tRNAAsp is modified to the (Q+) state before tRNAHis.

Animals↗
PubMed1981

Presence of queuine in Drosophila melanogaster: correlation of free pool with queuosine content of tRNA and effect of mutations in pteridine metabolism.

Queuine, a modified form of 7-deazaguanine present in certain transfer RNAs, is shown to occur in Drosophila melanogaster adults in a free form and its concentration varies as a function of age, nutrition and genotype. In several, but not all mutant strains, the concentrations of queuine and the Q(+) (queuine-containing) form of tRNATyr are correlated. The bioassay employs L-M cells which respond to the presence of queuine by an increase in their Q(+)tRNAAsp that is accompanied by a decrease in the Q(-)tRNAAsp isoacceptors. The increase in Q(+)tRNATyr in Drosophila that occurs on a yeast diet is accompanied by an increase in queuine. Similarly the increase of Q(+)tRNAs with age also is accompanied by an increase in free queuine. In two mutants, brown and sepia, these correlations were either diminished or failed to occur. Indeed, the extract of both mutants inhibited the response of the L-M cells to authentic queuine. When the pteridines that occur at abnormally high levels in sepia were used at 1 x 10(-6)M, the inhibition of the L-M cell assay occurred in the order biopterin greater than pterin greater than sepiapterin. These pteridines were also inhibitory for the purified guanine:tRNA transglycosylase from rabbit but the relative effectiveness then was pterin greater than biopterin greater than sepiapterin. Pterin was competitive with guanine in the enzyme reaction with Ki = 0.9 x 10(-7)M. Also when an extract of sepia was chromatographed on Sephadex G-50, the pteridine-containing fractions only were inhibitory toward the L-M cell assay or the enzyme assay. These results indicate that free queuine occurs in Drosophila but also that certain pteridines may interfere with the incorporation of queuine into RNA.

Animals↗
PubMed1980

Specific changes in Q-ribonucleoside containing transfer RNA species during Friend leukemia cell erythroid differentiation.

Changes in specific tRNA isoacceptors during Friend leukemia cell (F.L.C.) erythroid differentiation have been found to be concomitant with differences in the extent of the Q-base modification in certain species of tRNA. Transfer RNA was isolated from F.L.C. cultures after 0, 36, 48, 72, and 96 hr of DMSO induced differentiation. Changes in 17 isoacceptors of tRNAasn, tRNAasp, tRNAhis and tRNAtyr were compared by RPC-5 chromatography. Isoacceptors of these tRNA changed in relative amounts, following consistent trends throughout cell differentiation. The amount and distribution of Q-base containing tRNA isoacceptors was assayed by measuring the quanine-tRNA transferase catalyzed incorporation of [3H]-labeled guanine into tRNA species undermodified in Q-base followed by RPC-5 chormatography of the tRNA. The amount of Q-base containing tRNA species decreased in the first 48 hr after the induction, then increased again, indicating the level of Q-modification is correlated to the process of differentiation. Isoacceptors that lacked the Q-base were eluted late from RPC-5.

Animals↗
PubMed1980

Effect of diet on the queuosine family of tRNAs of germ-free mice.

The transfer RNAs for aspartic acid, asparagine, histidine, and tyrosine respond to codons in the third column of the genetic code and contain a hypermodified nucleoside known as queuosine (Q) in the first position of the anticodon of the major isoacceptor tRNA. Nothing is known about the physiological or biochemical function of Q. Germ-free mice were maintained for a period of nine tRNA half-lives on a chemically defined diet known to contain all essential constituents of the rodent diet but no Q or its base, queuine. The tRNAs for histidine and asparagine contained only 15% of the Q-containing isoacceptor tRNA. On the other hand, the Q-containing isoacceptor comprised 88% of the tRNAHis and 85% of the tRNAAsn in conventional mice and germ-free mice fed commercial mouse chow. Transfer RNAAsp and tRNATyr were completely modified with respect to Q in germ-free mice maintained on the chemically defined diet as well as on normal mouse chow. Germ-free mice fed the chemically defined diet contained normal amounts of the hypermodified base wye in tRNAPhe.

Animals↗
PubMed1979

Effects of plumbous ion on guanine metabolism.

The enzyme guanine aminohydrolase (guanase) is inhibited by low levels of Pb2+. The inhibition is noncompetitive and the Ki is 3.0 X 10(-6) M. The only other heavy metals that are inhibitory at low concentrations are Ag+, which is 36% more, and Hg2+, which is about 50% less inhibitory than Pb2+. The inhibition of guanase by Pb2+ and Hg2+ is synergistic and the inhibition of the enzyme was readily reversed by EDTA. The relationship of these studies with guanase and to the etiology and treatment of saturnine gout, which appears in humans suffering from lead poisoning, is discussed.

Aminohydrolases↗
PubMed1979

A factor in serum and amniotic fluid is a substrate for the tRNA-modifying enzyme tRNA-guanine transferase.

Q factor, a substance found in animal serum that enables cultured mammalian cells (L-M) to produce tRNA containing queuine (the base of "nucleoside Q", queuosine), has been purified to homogeneity from bovine amniotic fluid. Q factor causes the appearance of Q-containing tRNAAsp in the L-M cells cultivated in serum-free medium, and this was used as an assay to monitor the purification of Q factor. Q factor is a competitive inhibitor of guanine for rabbit reticulocyte tRNA-guanine trnsferase, with a K1 of 4.5 x 10(-8) M. Q factor is inactivated in both the L-M cell and tRNA-guanine transferase assays by treatment with periodate or cyanogen bromide, both of which react with queuine. In L-M cells, nearly complete conversion of Q-free to Q-containing tRNAAsp is observed within 24 hr after addition of pure Q factor to the medium; actinomycin D, cycloheximide, and cycloleucine, inhibitors of RNA synthesis, protein synthesis, and nucleic acid methylation, respectively, do not inhibit this conversion. The product of the reaction, catalyzed by pure rabbit reticulocyte tRNA-guanine transferase, between Q factor and rabbit reticulocyte Q-free tRNAHis is chromatographyically indistinguishable from Q-containing tRNAHis.

Amniotic Fluid↗
PubMed1978

Studies with a homogeneous enzyme from rabbit erythrocytes catalyzing the insertion of guanine into tRNA.

An enzyme that catalyzes a post-transcriptional modification of tRNA, resulting in replacement of a base from tRNA by guanine, has been purified 2600-fold from rabbit erythrocyte cytosol. The purest preparation migrates as a single protein band on polycrylamide gel electrophoresis and the enzymatic activity co-electrophoreses with this protein. The native enzyme has a molecular weight of 104,000 and is dissociated into two subunits of Mr= 60,000 and 43,000. The Km for guanine is 1.5 x 10(-7) M and for a pure guanine-accepting tRNA is 3.3 x 10(-9) M. The amino acid composition of the pure enzyme has been determined. To our knowledge this is the first study in which the molecular characteristics of a pure enzyme capable of modifying an internal position in tRNA has been reported.

Amino Acids↗
PubMed1978

Saturnine gout: lead-induced formation of guanine crystals.

Intravenous injection of a sublethal dose of lead acetate into a domestic pig resulted in a 4.5-fold increase of guanine in the urine, indicating an impairment in the conversion of guanine to xanthine. This impairment is probably due to the inhibition of guanine aminohydrolase (guanase), since the activity of this enzyme is inhibited by Pb2+ (the inhibition constant being 3.0 X 10(-6)M). Postmortem histological examination revealed concretions of crystalline material in the epiphyseal plate of the femoral head. Extraction of the section containing the concretions showed that they were guanine. The relation of these findings to saturnine gout is discussed.

Animals↗
PubMed1976

Partial purification and properties of the reticulocyte guanylating enzyme.

The guanylating enzyme which catalyzes the insertion of a guanine residue into one of the isoacceping tRNAHis of rabbit reticulocytes has been purified approximately one-hundred fold. It is free of nuclease activity. The enzyme does not catalyze the replacement of inserted radioactive guanine by unlabeled guanine, indicating that the reaction is irreversible. We have separated the histidyl-tRNA of reticulocytes into three isoacceptors. Previous work showed that the last histidyl-tRNA to elute from RPC-5 columns was the product of the guanylation reaction. This reports shows that the same late-eluting peak also contains the substrate for the guanylating enzyme, indicating that the guanine insertion reaction is chromatographically silent. The isoaccepting tRNAHis that is the substrate for the guanylating enzyme does not contain the hypermodified base known as Q. It is the other major reticulocyte tRNAHis that coantains Q, showing that at least in the reticulocyte the role of the guanylating enzyme is not the conversion of the Q form of tRNA to the homogeneic G form. The purified enzyme does not insert any base other than guanine into tRNA.

Animals↗
PubMed1976

Identification of the minor guanylated tRNA of rabbit reticulocytes.

Two of the tRNA's found in rabbit reticulocytes are substrates for a post-transcriptional modification leading to the incorporation of guanine into the polynucleotide chain. The major guanylated tRNA was previously identified as tRNA (His). In the present report we show that the minor guanylated tRNA is tRNA (Asn), and that just as in the case of tRNA (His), the guanine is located in an internal position. There are only two tRNA (Asn) in reticulocytes. We further show that one of these, the one that is not labeled with guanine, contains the hypermodified base known as Q. tRNA (Asn) does not contain Q.

Animals↗
PubMed1975

Guanine aminohydrolase in rat and mouse red cells: a potent inhibitor of guanylation of tRNA.

1. The red blood cells of mice and rats contained guanine aminohydrolase (EC 3.5.4.3). This enzyme was not present in rabbit, sheep or human red blood cells. 2. The enzyme from rat blood cells was separated into two activities by column chromatography on DEAE-cellulose. Both isozymes were labile but it was possible to show that the more abundant enzyme followed Michaelis-Menten kinetics, had an apparent Km of 4.0-10-6 M and was not activated by GTP nor inhibited by allantoin. 3. We believe, therefore, that guanine aminohydrolase was the protein in rat and mouse red blood cells that inhibited the enzyme (in rabbit reticulocytes) responsible for guanylation of tRNA.

Aminohydrolases↗