Biomedical subjects

A Billich

Publications and source records attributed to A Billich.

45 records · Page 3Linked to original sources

PubMed1988

Destruction of ethidium bromide in solution by ozonolysis.

Ethidium bromide can be rapidly destroyed in aqueous solutions or in isoamyl alcohol by ozonolysis in the presence of H2O2 to give a mixture of organic acids. In a variety of buffers commonly used in recombinant DNA technology destruction of ethidium bromide was more than 99.9%. The yellow reaction mixture after ozonolysis was shown to be nonmutagenic. This method may be used in laboratories for the disposal of ethidium bromide wastes.

Chemical Phenomena↗

PubMed1987

Enzymatic synthesis of cyclosporin A.

An enzyme fraction, isolated from crude extracts of the fungus Tolypocladium inflatum, strain 7939/F, is able to synthesize the undecapeptide cyclosporin A. The formation of cyclosporin A was monitored by incorporation of the radiolabeled constituent amino acids of cyclosporin A or by using S-adenosyl-L-[14C-methyl] methionine. The structure of cyclosporin A, synthesized enzymatically in vitro, was confirmed by chromatographic comparison with the authentic compound and by amino acid analyses. Replacement of L-2-aminobutyric acid in the reaction mixture by L-alanine, L-threonine, L-valine, or L-norvaline yields the naturally occurring cyclosporins B, C, D, and G. Also, D-alanine could be replaced by D-serine to yield [D-Ser8]cyclosporin A.

Amino Acids↗

PubMed1987

Monoclonal antibodies to the multienzyme enniatin synthetase. Production and use in structural studies.

Monoclonal antibodies have been prepared against the multifunctional enzyme enniatin synthetase, which catalyses the biosynthesis of the cyclodepsipeptide antibiotic enniatin. Five different antibodies (designated 1.56, 21.1, 25.91, 28.7 and 28.34) were characterized. 1.56, 21.1 and 25.91 were of IgG1 and 28.7 and 28.34 of IgM subclass. Binding studies showed that 21.1 and 25.91 are obviously directed against determinants based on the primary structure of the enzyme, whereas 28.7, 28.34 and 1.56 bind to the native enzyme. All antibodies inhibited enniatin formation. Based on their ability to inhibit different partial reactions of the multienzyme the antibodies could be divided into three groups: 21.1 and 25.91 inhibit valyl thioester formation, 1.56 additionally inhibits D-2-hydroxyisovaleric acid thioesterification, and 28.7 and 28.34 block both thioester sites as well as the N-methylation step. None of the antibodies affected the formation of L-valyl or D-hydroxyisovaleryl adenylate by the enzyme. The results indicate that there must be distinct thioester activation sites for valine and D-hydroxyisovalerate close to each other and in the neighbourhood of the methyltransferase site. The adenylation sites for D-hydroxy-isovalerate and L-valine are obviously located at some distance.

Affinity Labels↗

PubMed1986

Nucleoside phosphotransferase from malt sprouts. I. Isolation, characterization and specificity of the enzyme.

The nucleoside phosphotransferase of barley was isolated in four steps from dried malt sprouts and was obtained homogeneous according to several criteria. The enzyme transfers the phospho group from the 3'- or 5'-position of a nucleotide to the 5'-OH group of another nucleoside. Activated phosphomonoesters (e.g. 4-nitrophenyl phosphate) can be used as donors as well. In the absence of nucleosides the enzyme shows pronounced phosphatase activity towards 3'- and 5'-nucleotides as well as towards activated phosphomonoesters. 2'-Nucleotides are not hydrolysed. Studies of the phosphatase activity with a number of modified nucleotides demonstrate that the nucleotide substrates are bound by the phosphate group and by the base. The base can be extensively modified compared to naturally occurring nucleobases. Obviously, only a hydrophobic interaction with the enzyme is required; base-specific hydrogen bonds are of no importance. In the transferase reaction the acceptor nucleoside interacts only with the base which has a much more restricted specificity. Nucleotides which are donors do not need to be acceptors when they are nucleosides, while all acceptor nucleosides are efficient donors. Due to the pre-orientation of the base, the phosphate groups of the 3'- and 5'-nucleotides are in different positions relative to the groups of the enzyme that are involved in the catalysis. This is reflected in the kinetic parameters which are interpreted on the basis of a mechanism with a phospho-intermediate as postulated for the enzyme of Ives et al. [(1979) J. Biol. Chem. 254, 4339; (1982) J. Biol. Chem. 257, 4931]. Evidence is obtained that Km represents the ratio of the rate constants for the decay and the formation of the intermediate and that the kcat/Km-value can be used as indicator for the formation step.

Cyanogen Bromide↗

PubMed1986

Nucleoside phosphotransferase from malt sprouts. II. Studies on the active site and the phospho-intermediate.

The phospho-intermediate formed in the reaction of the nucleoside phosphotransferase is an acyl phosphate, the phosphorus bound to the gamma-carboxylate group of a glutamic acid. Reduction of this intermediate with sodium cyanoborotritiide yields labeled 2-amino-5-hydroxyvaleric acid after hydrolysis of the protein. Nucleophilic trapping of the intermediate with hydroxylamine during the reaction with substrates leads to N-phosphohydroxylamine, which is the only reaction product at a higher concentration of hydroxylamine. Evidence is obtained from modification experiments that in addition to the carboxylate group a histidine is involved in the reaction. The pKa-value for the histidine derived from the photoinactivation of the enzyme is 7.6, indicating that this group forms a salt bridge to a carboxylate group, probably that group attacking the phosphorus. The acceptor nucleosides are bound only by hydrophobic interactions of the base, a conclusion obtained from fluorescence studies and quenching experiments. The hydrophobic interaction obviously does not involve pi-interactions to tyrosine and tryptophan residues, since their fluorescence is not affected by addition of nucleotide inhibitors. Modification of these residues leads only to unspecific inactivation. From the Scatchard plot of the titration of the enzyme with 1,N6-ethenoadenosine 5'-phosphoramidate, an efficient inhibitor (Kd = 1.2 X 10(-5) M), it can be concluded that there is only one binding site on the dimeric enzyme.

Binding Sites↗

PubMed1986

Nucleoside phosphotransferase from malt sprouts. III. Studies on metal ion requirement, solvent effects, kinetics and reaction mechanism.

The nucleoside phosphotransferase from malt sprouts contains one Mg2 per dimeric enzyme molecule. This cation can be removed by EDTA, while other chelating agents are less efficient. The metal-free apoenzyme is inactive. Activity can be restored to its initial value by Mg2 or Co2 and to a minor extent by Mn2, Zn2, Cu2, Ni2 and Fe2. Thermal stability is reduced when the metal is removed but can be restored by addition of Mg2. Adenosine 3',5'-phosphate (cAMP) and arsenate, strong competitive inhibitors, reduce the rate of inactivation by EDTA considerably but do not reduce the rate for the reconstitution with Mg2. We therefore conclude that the phosphate group interacts electrostatically with Mg2 and that the inhibitor is not bound to the apoenzyme. The Sp-isomer of adenosine 3',5'-thionophosphate is a hundred times stronger inhibitor than the Rp-isomer and ten times stronger than cAMP; this allowed us to determine the relative position of the Mg2 in the active site. The imidazolium cation, previously detected as an essential part of the active site, obviously forms a salt bridge to the carboxylate group which attacks the phosphorus opposite to the leaving alcohol group. This conclusion is derived from the fact that organic solvents increase the rate of formation of the phospho-intermediate considerably, while higher concentrations of salt decrease it strongly. In addition, the imidazolium cation seems to polarize the P = O bond and to stabilize the negative charge at the phosphoryl oxygen in the pentacoordinate transition state; this is followed from the pH-dependence of the hydrolysis reaction. The kinetic results reveal that Km does not represent a binding equilibrium, but is the ratio of the rates for decay and formation of the covalent intermediate, while kcat/Km is an indicator for the formation step of the acyl phosphate. On the basis of all these informations it should be allowed to formulate a reaction mechanism, in which all steps of the transferase reaction have to be microscopically reversible.

Apoenzymes↗

PubMed1983

Synthesis, conformation and enzymatic properties of 1-(beta-D-allofuranosyl)uracil and some derivatives.

A new route for the synthesis of 1-(beta-D-allofuranosyl)uracil ("allo-uridine") and the corresponding 6'-deoxy-derivative ("6'-deoxy-allo-uridine") as well as for 1-(beta-D-altrofuranosyl) uracil ("altro-uridine") is described. NMR studies of allo-uridine revealed a preferred conformation with the base in anti-position, C-2'-endo-pucker of the sugar moiety, the 5'-OH-group above the furanose ring and the 5'-CH2OH-group in a gt position with the OH-group in the plane of the furanose ring. The same conformation is found for the 5'- and 6'-phosphate, indicated by the influence of the phosphate group on the H-6 signal. Allo-uridine is phosphorylated by the phosphotransferases from carrot and from malt sprouts only in the 6'-position. The phosphate ester is hydrolysed by unspecific phosphatases but not by 5'-nucleotidase. A (3' leads to 6')-dinucleoside phosphate is formed by pancreatic ribonuclease with 2',3'-cyclic cytidylic acid and allo-uridine. It is split by nuclease S1, but not by snake-venom phosphodiesterase. It has no primer activity for polynucleotide phosphorylase. All-uridine 6'-diphosphate could not be prepared enzymatically by nucleotide kinase or by chemical methods, where 5',6'-cyclic phosphates are formed, which are hydrolysed exclusively to 6'-monophosphates.

Indicators and Reagents↗

PubMedDate not supplied

Stimulation of MCF-7 breast cancer cell proliferation by estrone sulfate and dehydroepiandrosterone sulfate: inhibition by novel non-steroidal steroid sulfatase inhibitors.

Steroid sulfatase (STS) regulates the formation of active steroids from systemic precursors, such as estrone sulfate and dehydroepiandrosterone sulfate (DHEAS). In breast tissues, this pathway is a source for local production of estrogens, which support the growth of endocrine-dependent tumours. Therefore, inhibitors of STS could have therapeutic potential. In this study, we report on substituted chromenone sulfamates as a novel class of non-steroidal irreversible inhibitors of STS. The compounds are substantially more potent (6- to 80-fold) than previously described types of non-steroidal inhibitors when tested against purified STS. In MCF-7 breast cancer cells, they inhibit STS activity with IC(50) below 100 pM. Importantly, the compounds also potently block estrone sulfate-stimulated growth of MCF-7 cells, again with IC(50) below 100 pM. For one compound, we also observed a lack of any estrogenic effect at high concentrations (1 microM). We also demonstrate for the first time that STS inhibitors can block the DHEAS-stimulated growth of MCF-7 cells. Interestingly, this cannot be achieved with specific inhibitors of the aromatase, suggesting that stimulation of MCF-7 cell growth by DHEAS follows an aromatase-independent pathway. This gives further justification to consider steroid sulfatase inhibitors as potential drugs in the therapy of breast cancer.

Arylsulfatases↗

PubMedDate not supplied

Colorimetric assay of HIV-1 proteinase suitable for high-capacity screening.

The cleavage of substrate peptides by HIV-1 proteinase can be monitored by reaction of exposed amino groups with 2,4,6-trinitrobenzenesulfonic acid to yield yellow-colored products. This method provides a high-capacity assay of the enzyme which can be performed on microtiter plates.

Amino Acid Sequence↗