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F Ruzicka

Publications and source records attributed to F Ruzicka.

At least 19 recordsLinked to original sources
PubMed1990

Remodeling hexose-1-phosphate uridylyltransferase: mechanism-inspired mutation into a new enzyme, UDP-hexose synthase.

Hexose-1-phosphate uridylyltransferase catalyzes the interconversion of UDP-galactose and glucose-1-P with UDP-glucose and galactose-1-P by a double-displacement mechanism through a covalent intermediate (E-UMP), in which UMP is bonded to one of two histidine residues at the active site, H164 or H166. To identify which histidine is the nucleophilic catalyst, we prepared two specific mutants of the enzyme from Escherichia coli, H164G and H166G, in each of which the imidazole ring and methylene carbon of one histidine are deleted. To determine whether the function of the deleted imidazole in these mutants could be carried out by the imidazole ring in uridine 5'-(phosphoimidazolate) (UMP-Im), we examined the mutant proteins for catalytic activity in the reaction of UMP-Im with glucose-1-P to form UDP-glucose and imidazole. The mutant H166G catalyzes this reaction, as well as the reverse reaction, by a sequential kinetic mechanism involving ternary complexes as intermediates. The mutant enzyme also accepts galactose-1-P as a substrate to form UDP-galactose. Hexose-1-P uridylyltransferase does not catalyze these reactions, and H166G does not catalyze the wild-type reaction. The substrate Km values for the mutant enzyme are similar to those for hexose-1-P uridylyltransferase. The value of kcat in the direction of UDP-glucose formation is 1.31 +/- 0.01 s-1, compared with 350 s-1 for hexose-1-P uridylyltransferase, and in the reverse direction kcat is 4.8 +/- 0.4 s-1, compared with 960 s-1 for the wild-type enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)

Glucosephosphates↗
PubMed1990

Methane monooxygenase catalyzed oxygenation of 1,1-dimethylcyclopropane. Evidence for radical and carbocationic intermediates.

Methane monooxygenase catalyzes the oxygenation of 1,1-dimethylcyclopropane in the presence of O2 and NADH to (1-methylcyclopropyl)methanol (81%), 3-methyl-3-buten-1-ol (6%), and 1-methyl-cyclobutanol (13%). Oxygenation by 18O2 using the purified enzyme proceeds with incorporation of 18O into the products. Inasmuch as methane monooxygenase catalyzes the insertion of O from O2 into a carbon-hydrogen bond of alkanes, (1-methylcyclopropyl)methanol appears to be a conventional oxygenation product. 3-Methyl-3-buten-1-ol is a rearrangement product that can be rationalized on the basis that enzymatic oxygenation of 1,1-dimethylcyclopropane proceeds via the (1-methylcyclopropyl)carbinyl radical, which is expected to undergo rearrangement with ring opening to the homoallylic 3-methyl-3-buten-1-yl radical in competition with conventional oxygenation. Oxygenation of the latter radical gives 3-methyl-3-buten-1-ol. 1-Methylcyclobutanol is a ring-expansion product, whose formation is best explained on the basis that the 1-methylcyclobutyl tertiary carbocation is an oxygenation intermediate. This cation would result from rearrangements of carbocations derived by one-electron oxidation of either radical intermediate. The fact that both 3-methyl-3-buten-1-ol and 1-methylcyclobutanol are produced suggests that the oxygenation mechanism involves both radical and carbocationic intermediates. Radicals and carbocations can both be intermediates if they are connected by an electron-transfer step. A reasonable reaction sequence is one in which the cofactor (mu-oxo)diiron reacts with O2 and two electrons to generate a hydrogen atom abstracting species and an oxidizing agent. The hydrogen-abstracting species might be the enzymic radical or another species generated by the iron complex and O2.(ABSTRACT TRUNCATED AT 250 WORDS)

Cyclopropanes↗
PubMed1990

Growth inhibitory effects of interferon-beta but not interferon-alpha on human glioma cells: correlation of receptor binding, 2',5'-oligoadenylate synthetase and protein kinase activity.

The antiproliferative effects of human recombinant interferon-alpha (rIFN-alpha A) and interferon-beta (rIFN-beta ser) were assessed in vitro against seven human glioma cell lines. Further analysis of one of these lines (EFC-2) in response to rIFN-alpha A demonstrated a minimum growth inhibition by day 6 of treatment, whereas a 50% inhibition of cell growth was observed with a dose of 50 U/ml of IFN-beta ser. No significant growth inhibition was seen by rIFN-alpha A at doses up to 500 U/ml. Addition of rIFN-alpha A to rIFN-beta ser-treated EFC-2 cells neither suppressed nor augmented the antiproliferative response to IFN-beta ser. The binding of 125I-labeled rIFN-alpha A or 125I-labeled rIFN-beta ser to EFC-2 cells was inhibited competitively by increasing concentrations of either unlabeled rIFN-alpha A or rIFN-beta ser. This suggests that the cellular receptors for both rIFN-alpha A and rIFN-beta ser appear to be intact and appear to bind both agents equally. Furthermore, incubation of EFC-2 cells for 72 h with either rIFN-alpha A or rIFN-beta ser resulted in an increase in 2',5'-oligoadenylate (2-5A) synthetase activity 5-fold with rIFN-alpha A and 50-fold with rIFN-beta ser. Similarly, the 68-kD IFN-induced protein kinase was induced substantially with rIFN-beta ser but only slightly induced with rIFN-alpha A treatment. These results suggest that EFC-2 human glioma cells demonstrate a differential sensitivity in terms of growth inhibition to rIFN-beta ser and to rIFN-alpha A which appears to correlate with a differential induction of both intracellular 2-5A synthetase and protein kinase activity. These results cannot be explained solely on the basis of surface receptor binding of rIFN-alpha A and rIFN-beta ser. These data do suggest that, for human glioma cells in culture, type I IFN receptors may display a subtle architectural variation that allows equivalent binding of both IFN-alpha and IFN-beta ser, but allows an enhanced signal transduction and biological effect only after binding a specific IFN subtype.

2',5'-Oligoadenylate Synthetase↗
PubMed1977

[New findings on the structure of chromosomes (author's transl)].

The structural element of eukaryotic chromosomes is the chromatin fibre consisting of histones and DNA. The chromatin fibre is about 100--200 A thick. One chromatid is built up from one chromatin fibre running through from one end to the other and laid in numerous irregular foldings. The chromatin fibre is a chain of nucleosomes. These are globular histone bodies around which the DNA winds. Nucleosomes can be observed in isolated chromatin fibrils as well as in thin sections of chromosomes after different modes of fixation. Prophasic chromosomes or early premature condensed chromosomes are thin uncoiled threads. With chromosome condensation a major coiling is seen. No constant regular arrangement of the chromatin fibril besides the major coils is observed. A rather diffuse decondensation takes place in ana- and telophase.

Animals↗
PubMed1976

Fine structure of 33 258 H-treated chromosomes.

Metaphase chromosomes of mouse strain L cells show strikingly uncondensed pericentric heterochromatic regions after treatment of living cells with the benzimidazol-derivative 33 258 Hoechst. In electron micrographs of total preparations after G-band staining the chromosomes are seen to be made up of irregularly folded fibrils of 200-400 A in diameter. In the uncondensed regions only very few fibrils laid loose loops are present, making it probable that only one fibril forms one chromatid.

Animals↗
PubMed1976

[3 cases of eosinophilic leukemia with atypical granulation in the eosinophils and neutrophils].

Three patients with eosinophilic leukemia and atypical granulation in the eosinophils are described. A remarkable light and electron microscopic finding was the appearance of enormous granules in the neurtophils and eosinophils. In addition the usual anomalies seen in acute leukemia, for example asynchronisation in cell maturation, fibrillar bundles, shrinkage of nucleolus and cell organelles, signs of degeneration and also the auer-rods characteristic of AML are observed.

Acute Disease↗
PubMed1975

A case of acute lymphoblastic leukemia and thorotrast-accumulation.

A case of acute lymphoblastic leukemia (ALL), in a 50 years old patient, 26 years after thorotrast injection, is reported. In spite of intensive therapy, he died 2 years after diagnosis of disease. The cytogenetic showed the usual thorotrast radiation-induced abnormalities, although to a greater extent than reported in leterature. Furthermore a hypodiploidy was present, which was connected with ALL. In addition the patient exhibited the interesting phenomenon of giant satellites on one of his D14-chromosomes. This abnormally was found also in his mother and son. The question arises, how far the inherited cytogenetic pattern and the thorotrast radiation each contributed to the development of ALL.

Bone Marrow Examination↗
PubMed1975

[Spontaneous radial segmentation of the nuclei of myeloblasts in a case of chronic granulocytic leukemia during blastic crisis. an electromicroscopical study (author's transl)].

By means of electron microscopy, myeloblasts with spontaneous radial segmentation of the nuclei from a patient with micromegakaryotic chronic myelosis in the blastic crisis were analysed. The centriol was found in the center of the cells and microtubules were found between the segments of the nucleus. These observations support the hypothesis that spontaneous radial segmentations of the nuclei of tumor cells is associated with a contractile process of the microtubules with split the nucleus.

Adult↗